Abstract:

Nucleic acids encoding a new family of chemokines, the CX3C family, from a
mammal, reagents related thereto, including specific antibodies, and
purified proteins are described. Methods of using said reagents and
related diagnostic kits are also provided.

Claims:

1. A method for treating inflammation, which is mediated by
chemoattraction of peripheral blood monocytes or T-cells, in a subject
comprising administering, to said subject, an effective dose of an
isolated antibody or antigen-binding fragment thereof, or a
pharmaceutical composition thereof comprising said antibody or fragment
and a pharmaceutically acceptable carrier, which antibody or fragment
specifically binds to a polypeptide wherein said polypeptidea)
chemoattracts peripheral blood monocytes or T-cells,b) consists of an
amino acid sequence comprising at least 95% sequence identity to residues
25 to 397 of the amino acid sequence set forth in SEQ ID NO: 4, andc)
comprises a chemokine domain identical to residues 25 to 100 of the amino
acid sequence set forth in SEQ ID NO: 4.

2. The method of claim 1 wherein said isolated antibody or antigen-binding
fragment thereof specifically binds to a polypeptide consisting of an
amino acid sequence set forth in SEQ ID NO: 4.

3. The method of claim 1 wherein said isolated antibody or antigen-binding
fragment thereof specifically binds to a polypeptide consisting of Gln25
to Gly100 of the amino acid sequence set forth in SEQ ID NO: 4.

4. The method of claim 1 wherein said polypeptide is denatured.

5. The method of claim 3 wherein said isolated antibody or antigen-binding
fragment thereof is produced in a process comprising immunizing an animal
with a purified or recombinantly produced polypeptide comprising Gln25 to
Gly100 of the amino acid sequence set forth in SEQ ID NO: 4.

6. The method of claim 5 wherein said isolated antibody or antigen-binding
fragment thereof is produced in a process comprising immunizing an animal
with a purified or recombinantly produced polypeptide comprising a mature
polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

7. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is detectably labeled.

8. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is an antibody and the antibody is a polyclonal antibody.

9. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is an antibody and the antibody is a monoclonal antibody.

10. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is a fragment and the fragment is a Fab.

11. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is a fragment and the fragment is a F(ab).sub.2.

12. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof is sterile.

13. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof exhibits a KD to said polypeptide of greater than about 100
nM.

14. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof exhibits a KD to said polypeptide of greater than about 30
nM.

15. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof exhibits a KD to said polypeptide of greater than about 10
nM.

16. The method of claim 1 wherein the antibody or antigen-binding fragment
thereof exhibits a KD to said polypeptide of greater than about 3
nM.

17. A method for treating inflammation, which is mediated by
chemoattraction of peripheral blood monocytes or T-cells, in a subject
comprising administering, to said subject, an effective dose of a
monoclonal antibody that binds specifically to a polypeptide consisting
of amino acids 25-397 of SEQ ID NO: 4, or a pharmaceutical composition
thereof which comprises a pharmaceutically acceptable carrier.

18. The method of claim 1 for treating inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells, in a subject
comprising administering, to said subject, an effective dose of a
pharmaceutical composition comprising the antibody or antigen-binding
fragment and a pharmaceutically acceptable carrier.

19. A method for treating inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells, in a subject
comprising administering, to said subject, an effective dose of a
pharmaceutical composition comprising a monoclonal antibody that binds
specifically to a polypeptide consisting of the amino acid sequence set
forth in SEQ ID NO: 4 and a pharmaceutically acceptable carrier.

20. The method of claim 1 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is arthritis.

21. The method of claim 1 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is multiple
sclerosis.

22. The method of claim 1 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is neurogenic
inflammation.

23. The method of claim 17 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is arthritis.

24. The method of claim 17 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is multiple
sclerosis.

25. The method of claim 17 wherein the inflammation which is mediated by
chemoattraction of peripheral blood monocytes or T-cells is neurogenic
inflammation.

Description:

[0001]The present application is a continuation of U.S. patent application
Ser. No. 11/495,801, filed Jul. 28, 2006; which is a continuation of U.S.
patent application Ser. No. 10/397,559, filed Mar. 25, 2003; which is a
division of U.S. patent application Ser. No. 09/771,023, filed Jan. 25,
2001, now U.S. Pat. No. 6,566,503; which is a division of U.S. patent
application Ser. No. 09/093,482, filed Jun. 8, 1998; which is a division
of U.S. patent application Ser. No. 08/786,068, filed Jan. 21, 1997;
which is a continuation of U.S. patent application Ser. No. 08/649,006,
filed May 16, 1996, now U.S. Pat. No. 6,548,654; which is a continuation
of U.S. patent application Ser. No. 08/590,828 filed Jan. 24, 1996, each
of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002]The present invention contemplates compositions related to proteins
which function in controlling development, differentiation, trafficking,
and physiology of mammalian cells, e.g., cells of a mammalian immune
system. In particular, it provides proteins which regulate or evidence
development, differentiation, and function of various cell types,
including hematopoietic cells.

[0004]For some time, it has been known that the mammalian immune response
is based on a series of complex cellular interactions, called the "immune
network." Recent research has provided new insights into the inner
workings of this network. While it remains clear that much of the
response does, in fact, revolve around the network-like interactions of
lymphocytes, macrophages, granulocytes, and other cells, immunologists
now generally hold the opinion that soluble proteins, known as
lymphokines, cytokines, or monokines, play a critical role in controlling
these cellular interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell modulatory
factors, an understanding of which should lead to significant
advancements in the diagnosis and therapy of numerous medical
abnormalities, e.g., immune system and other disorders.

[0005]Lymphokines apparently mediate cellular activities in a variety of
ways. They have been shown to support the proliferation, growth, and
differentiation of pluripotential hematopoietic stem cells into vast
numbers of progenitors comprising diverse cellular lineages making up a
complex immune system. These interactions between cellular components are
necessary for a healthy immune response. These different cellular
lineages often respond in a different manner when lymphokines are
administered in conjunction with other agents.

[0006]The chemokines are a large and diverse superfamily of proteins. The
superfamily is subdivided into three branches, based upon whether the
first two cysteines in the classical chemokine motif are adjacent (termed
the "C--C" branch) or spaced by an intervening residue ("C--X--C"), or a
new branch which lacks two cysteines in the corresponding motif,
represented by the chemokines known as lymphotactins. See, e.g., Schall
and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and
Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.

[0007]Many factors have been identified which influence the
differentiation process of precursor cells, or regulate the physiology or
migration properties of specific cell types. These observations indicate
that other factors exist whose functions in immune function were
heretofore unrecognized. These factors provide for biological activities
whose spectra of effects may be distinct from known differentiation or
activation factors. The absence of knowledge about the structural,
biological, and physiological properties of the regulatory factors which
regulate cell physiology in vivo prevents the modification of the effects
of such factors. Thus, medical conditions where regulation of the
development or physiology of relevant cells is inappropriate remain
unmanageable.

SUMMARY OF THE INVENTION

[0008]The present invention reveals the existence of a previously unknown
class of chemokine-motif containing molecules which are hereby designated
the CX3C chemokines. The CX3Ckines have three amino acids which separate
the cysteines in the corresponding region of the chemokine motif. Based
on sequence analysis of the two CX3C protein sequences described below,
it is apparent that the CX3Ckines do not belong to the C, C--C, or
C--X--C chemokine families. They represent the first known members of a
new heretofore unidentified class of chemokines designated CX3Ckines, or
alternatively, the CX3C family of chemokines.

[0009]The present invention provides a composition of matter selected from
an antibody binding site which specifically binds to a mammalian CX3C
chemokine; an expression vector encoding a mammalian CX3C chemokine or
fragment thereof; a substantially pure protein which is specifically
recognized by the antibody binding site; and a substantially pure CX3C
chemokine or peptide thereof, or a fusion protein comprising a 30 amino
acid fragment of CX3C chemokine sequence.

[0010]In the antibody binding site embodiments, the antibody binding site
may be: specifically immunoreactive with a mature protein selected from
the group consisting of the polypeptides of SEQ ID NO: 2, 4, 6 and 8;
raised against a purified or recombinantly produced human or mouse CX3C
chemokine; in a monoclonal antibody, Fab, or F(ab)2; immunoreactive
with denatured antigen; or in a labeled antibody. In certain embodiments;
the antibody binding site is detected in a biological sample by a method
of: contacting a binding agent having an affinity for CX3C chemokine
protein with the biological sample; incubating the binding agent with the
biological sample to form a binding agent:CX3C chemokine protein complex;
and detecting the complex. In a preferred embodiment, the biological
sample is human, and the binding agent is an antibody.

[0011]A kit embodiment is provided possessing a composition, described
above, with either instructional material for the use of the composition;
or segregation of the composition into a container.

[0012]A nucleic acid embodiment of the invention includes an expression
vector encoding a CX3C chemokine protein, wherein the protein
specifically binds an antibody generated against an immunogen selected
from the mature polypeptide portions of SEQ ID NO: 2, 4, 6, and 8. The
vector may: encode a CX3C chemokine polypeptide with complete sequence
identity to a naturally occurring human CX3C chemokine domain; encode a
CX3C chemokine protein comprising sequence selected from the polypeptides
of SEQ ID NO: 2, 4, 6, and 8; or comprise sequence selected from the
nucleic acids of SEQ ID NO: 1, 3, 5, or 7. In other embodiments, the
vector is capable of selectively hybridizing to a nucleic acid encoding a
CX3C chemokine protein, e.g., a mature protein coding segment of SEQ ID
NO: 1, 3, 5, or 7. In various preferred embodiments, the isolated nucleic
acid is detected in a biological sample by a method: contacting a
biological sample with a nucleic acid probe capable of selectively
hybridizing to the nucleic acid; incubating the nucleic acid probe with
the biological sample to form a hybrid of the nucleic acid probe with
complementary nucleic acid sequences present in the biological sample;
and determining the extent of hybridization of the nucleic acid probe to
the complementary nucleic acid sequences. In such method, preferably the
nucleic acid probe is capable of hybridizing to a nucleic acid encoding a
protein consisting of the polypeptides of SEQ ID NO: 2, 4, 6, or 8.

[0013]In protein embodiments, the isolated CX3C chemokine protein will
preferably be of approximately 11,000 to 15,000 daltons when in
unglycosylated form, and the CX3C chemokine protein specifically binds to
an antibody generated against an immunogen; the polypeptides of SEQ ID
NO: 2, 4, 6, or 8; and the CX3C chemokine lacks the cysteine structural
motifs and sequence characteristic of a C, a CC, or a CXC chemokine. In
various embodiments, the isolated CX3C chemokine protein is: selected
from human CX3Ckine or mouse CX3Ckine; consists of a polypeptide
comprising sequence from SEQ ID NO: 2, 4, 6, or 8; recombinantly
produced, or a naturally occurring protein.

[0014]The present invention also embraces a cell transfected with the
nucleic acid encoding a CX3C chemokine, e.g., where the nucleic acid has
SEQ ID NO: 1, 3, 5, or 7.

[0015]The invention also provides a method of modulating physiology or
development of a cell by contacting the cell with a CX3C chemokine, or an
antagonist of the chemokine. In preferred embodiments, the physiology is
attraction, and the cell is a peripheral blood monocyte or a T cell.

[0017]The best characterized embodiment of this family of proteins was
discovered from a human and is designated human CX3C chemokine (GenBank
Accession No. H14940). See, SEQ ID NO: 1-4 An additional CX3Ckine,
represented by a mouse molecule, designated mouse CX3Ckine, is also
described herein. See Table 1 and SEQ ID NO: 5-8. The descriptions below
are directed, for exemplary purposes, to primate and rodent embodiments,
e.g., human and mouse, but are likewise applicable to related embodiments
from other, e.g., natural, sources. These sources should include various
vertebrates, typically warm blooded animals, e.g., birds and mammals,
particularly domestic animals, and primates.

[0018]In the human sequence (SEQ ID NO: 1-4), the signal sequence runs
from about Met1 to Gly24, thus the mature polypeptide begins at about
Gln25 and ends at about Val 397. A chemokine domain runs from about Gln25
to about Gly100; a stalk region, which possesses many potential
glycosylation sites, runs from about Gly101 to about Gln341; a
tranmembrane region begins at about Ala342 and ends at about Thr361; and
an intracellular domain, containing two tyrosine phosphorylation sites at
residues 382 and 392, runs from about Tyr362 to Val397.

[0019]The CX3Ckine proteins of this invention are defined in part by their
physicochemical and biological properties. The biological properties of
the human and mouse CX3Ckines described herein, e.g., human CX3Ckine and
mouse CX3Ckine, are defined by their amino acid sequence, and mature
size. They also should share biological properties. The human and mouse
CX3Ckine molecules exhibit about 70-80% amino acid identity, depending on
whether the signal or mature sequences are compared. One of skill will
readily recognize that some sequence variations may be tolerated, e.g.,
conservative substitutions or positions remote from the helical
structures, without altering significantly the biological activity of the
molecule.

[0022]The CX3C chemokine producing profile of various cells is elucidated
herein. Screening a cDNA library generated from brain provided a novel
cytokine, designated human CX3Ckine. Human CX3Ckine exhibits distant
similarity to members of the C, C--C, and C--X--C chemokine families,
with another heretofore unrecognized number of amino acid residues
separating the characteristic cysteines in the motif which is peculiar to
and partially defines chemokines. These observations suggest that the
CX3Ckines represent novel additions to the chemokine superfamily.

[0023]CX3C chemokine protein biochemistry was assessed in mammalian
expression systems. Human embryonic kidney 293 cells (HEK 293)
transfected with a mammalian expression construct encoding full-length
CX3C chemokine were metabolically labeled with 35S cysteine and
methionine. CX3C chemokine was produced as a protein of Mr ˜95 kDa;
control transfected supernatants contained no such species. Neuraminidase
and glycosidases reduced the Mr of CX3C chemokine from ˜95 kDa to
˜45 kDa, suggesting that the recombinant form, is glycosylated
substantially. CX3C chemokine cDNA, encoding a predicted membrane-bound
protein, encodes a glycoprotein which is released from cells by an
undefined mechanism.

[0024]The pro-migratory activities of CX3C chemokine have been assessed in
microchemotaxis assays. CX3C chemokine appears to be a potent attractant
of peripheral blood monocytes and T cells. Pro-migratory activity for
blood neutrophils has been difficult to demonstrate.

[0025]The CX3C chemokine gene has been mapped to human chromosome 16.
Mapping studies also indicate the possibility of a pseudogene or related
gene on human chromosome 14. Sequencing of genomic DNA fragments suggests
CX3C chemokine gene has an intron which begins near or in the codon
encoding Ile 64. Other intron/exon boundaries have yet to be mapped, but
such will be easily accomplished by standard methods.

[0026]The membrane bound form of CX3Ckine possesses proadherent properties
for circulating T cells and monocytes. A secreted or soluble form,
consisting of the chemokine domain and the stalk region, is able to
inhibit this proadhesive activity. This suggests that the membrane bound
form of CX3Ckine may be a potent regulator of circulating leukocytes, and
thus may be involved in various inflammatory diseases, e.g., arthritis.
The soluble form may be used as a regulator of proadherence, especially
in conditions of compromised immune response.

[0027]CX3C chemokine's properties as a T cell and monocyte
chemoattractant, coupled with its distribution in brain and other organs,
suggests that CX3C chemokine may be involved in the pathogenesis of such
CNS inflammatory disorders as multiple sclerosis, and other pathologies
involving neurogenic inflammation. Since CX3C chemokine distribution is
not limited to the brain, however, the entire spectrum of inflammatory,
infectious, and immunoregulatory states thought to involve other
chemokines must also now be considered for CX3C chemokine. See, e.g.,
Frank, Et al. (eds.) (1995) Samter's Immunologic Diseases 5th ed., vols.
I and II, Little, Brown, and Co., Boston, Mass.

II. Definitions

[0028]The term "binding composition" refers to molecules that bind with
specificity to a CX3Ckine, e.g., in an antibody-antigen interaction.
However, other compounds, e.g., receptor proteins, may also specifically
associate with CX3Ckines to the exclusion of other molecules. Typically,
the association will be in a natural physiologically relevant
protein-protein interaction, either covalent or non-covalent, and may
include members of a multiprotein complex, including carrier compounds or
dimerization partners. The molecule may be a polymer, or chemical
reagent. No implication as to whether a CX3Ckine is either the ligand or
the receptor of a ligand-receptor interaction is necessarily represented,
other than whether the interaction exhibits similar specificity, e.g.,
specific affinity. A functional analog may be a ligand with structural
modifications, or may be a wholly unrelated molecule, e.g., which has a
molecular shape which interacts with the appropriate ligand binding
determinants. The ligands may serve as agonists or antagonists of the
receptor, see, e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's:
The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press,
Tarrytown, N.Y.

[0029]The term "binding agent:CX3Ckine protein complex", as used herein,
refers to a complex of a binding agent and a CX3Ckine protein that is
formed by specific binding of the binding agent to the CX3Ckine protein,
e.g., preferably the chemokine domain. Specific binding of the binding
agent means that the binding agent has a specific binding site that
recognizes a site on the CX3Ckine protein. For example, antibodies raised
to a CX3Ckine protein and recognizing an epitope on the CX3Ckine protein
are capable of forming a binding agent:CX3Ckine protein complex by
specific binding. Typically, the formation of a binding agent:CX3Ckine
protein complex allows the measurement of CX3Ckine protein in a mixture
of other proteins and biologics. The term "antibody:CX3Ckine protein
complex" refers to an embodiment in which the binding agent is an
antibody. The antibody may be monoclonal, polyclonal, or a binding
fragment of an antibody, e.g, an Fab of F(ab)2 fragment. The
antibody will preferably be a polyclonal antibody for cross-reactivity
purposes.

[0030]"Homologous" nucleic acid sequences, when compared, exhibit
significant similarity. The standards for homology in nucleic acids are
either measures for homology generally used in the art by sequence
comparison and/or phylogenetic relationship, or based upon hybridization
conditions. Hybridization conditions are described in greater detail
below.

[0031]An "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or
a mixed polymer, which is substantially separated from other biologic
components which naturally accompany a native sequence, e.g., proteins
and flanking genomic sequences from the originating species. The term
embraces a nucleic acid sequence which has been removed from its
naturally occurring environment, and includes recombinant or cloned DNA
isolates and chemically synthesized analogs, or analogs biologically
synthesized by heterologous systems. A substantially pure molecule
includes isolated forms of the molecule. An isolated nucleic acid will
usually contain homogeneous nucleic acid molecules, but will, in some
embodiments, contain nucleic acids with minor sequence heterogeneity.
This heterogeneity is typically found at the polymer ends or portions not
critical to a desired biological function or activity.

[0032]As used herein, the term "CX3Ckine protein" shall encompass, when
used in a protein context, a protein having amino acid sequences,
particularly from the chemokine motif portions, shown in SEQ ID NO: 2, 4,
6, or 8, or a significant fragment of such a protein, e.g., preferably
the chemokine domain. The invention also embraces a polypeptide which
exhibits similar structure to human or mouse CX3Ckine, e.g., which
interacts with CX3Ckine specific binding components. These binding
components, e.g., antibodies, typically bind to a CX3Ckine with high
affinity, e.g., at least about 100 nM, usually better than about 30 nM,
preferably better than about 10 nM, and more preferably at better than
about 3 nM.

[0033]The term "polypeptide" or "protein" as used herein includes a
significant fragment or segment of chemokine motif portion of a CX3Ckine,
and encompasses a stretch of amino acid residues of at least about 8
amino acids, generally at least 10 amino acids, more generally at least
12 amino acids, often at least 14 amino acids, more often at least 16
amino acids, typically at least 18 amino acids, more typically at least
20 amino acids, usually at least 22 amino acids, more usually at least 24
amino acids, preferably at least 26 amino acids, more preferably at least
28 amino acids, and, in particularly preferred embodiments, at least
about 30 or more amino acids, e.g., 35, 40, 45, 50, 60, 70, 80, etc.

[0034]A "recombinant" nucleic acid is defined either by its method of
production or its structure. In reference to its method of production,
e.g., a product made by a process, the process is use of recombinant
nucleic acid techniques, e.g., involving human intervention in the
nucleotide sequence, typically selection or production. Alternatively, it
can be a nucleic acid made by generating a sequence comprising fusion of
two fragments which are not naturally contiguous to each other, but is
meant to exclude products of nature, e.g., naturally occurring mutants.
Thus, for example, products made by transforming cells with any
non-naturally occurring vector is encompassed, as are nucleic acids
comprising sequence derived using any synthetic oligonucleotide process.
Such is often done to replace a codon with a redundant codon encoding the
same or a conservative amino acid, while typically introducing or
removing a sequence recognition site. Alternatively, it is performed to
join together nucleic acid segments of desired functions to generate a
single genetic entity comprising a desired combination of functions not
found in the commonly available natural forms. Restriction enzyme
recognition sites are often the target of such artificial manipulations,
but other site specific targets, e.g., promoters, DNA replication sites,
regulation sequences, control sequences, or other useful features may be
incorporated by design. A similar concept is intended for a recombinant,
e.g., fusion, polypeptide. Specifically included are synthetic nucleic
acids which, by genetic code redundancy, encode polypeptides similar to
fragments of these antigens, and fusions of sequences from various
different species variants. Mutation of protease cleavage sites may also
be accomplished.

[0035]"Solubility" is reflected by sedimentation measured in Svedberg
units, which are a measure of the sedimentation velocity of a molecule
under particular conditions. The determination of the sedimentation
velocity was classically performed in an analytical ultracentrifuge, but
is typically now performed in a standard ultracentrifuge. See, Freifelder
(1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., San Francisco,
Calif.; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3,
W.H. Freeman & Co., San Francisco, Calif. As a crude determination, a
sample containing a putatively soluble polypeptide is spun in a standard
full sized ultracentrifuge at about 50K rpm for about 10 minutes, and
soluble molecules will remain in the supernatant. A soluble particle or
polypeptide will typically be less than about 30 S, more typically less
than about 15 S, usually less than about 10 S, more usually less than
about 6 S, and, in particular embodiments, preferably less than about 4
S, and more preferably less than about 3 S. Solubility of a polypeptide
or fragment depends upon the environment and the polypeptide. Many
parameters affect polypeptide solubility, including temperature,
electrolyte environment, size and molecular characteristics of the
polypeptide, and nature of the solvent. Typically, the temperature at
which the polypeptide is used ranges from about 4° C. to about
65° C. Usually the temperature at use is greater than about
18° C. and more usually greater than about 22° C. For
diagnostic purposes, the temperature will usually be about room
temperature or warmer, but less than the denaturation temperature of
components in the assay. For therapeutic purposes, the temperature will
usually be body temperature, typically about 37° C. for humans,
though under certain situations the temperature may be raised or lowered
in situ or in vitro.

[0036]The size and structure of the polypeptide should generally be
evaluated in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in a
quaternary structure, e.g., to confer solubility, or associated with
lipids or detergents in a manner which approximates natural lipid bilayer
interactions.

[0037]The solvent will usually be a biologically compatible buffer, of a
type used for preservation of biological activities, and will usually
approximate a physiological solvent. Usually the solvent will have a
neutral pH, typically between about 5 and 10, and preferably about 7.5.
On some occasions, a detergent will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate), or a low
enough concentration as to avoid significant disruption of structural or
physiological properties of the protein.

[0038]"Substantially pure" in a protein context typically means that the
protein is isolated from other contaminating proteins, nucleic acids, and
other biologicals derived from the original source organism. Purity, or
"isolation" may be assayed by standard methods, and will ordinarily be at
least about 50% pure, more ordinarily at least about 60% pure, generally
at least about 70% pure, more generally at least about 80% pure, often at
least about 85% pure, more often at least about 90% pure, preferably at
least about 95% pure, more preferably at least about 98% pure, and in
most preferred embodiments, at least 99% pure. Similar concepts apply,
e.g., to antibodies or nucleic acids.

[0039]"Substantial similarity" in the nucleic acid sequence comparison
context means either that the segments, or their complementary strands,
when compared, are identical when optimally aligned, with appropriate
nucleotide insertions or deletions, in at least about 50% of the
nucleotides, generally at least 56%, more generally at least 59%,
ordinarily at least 62%, more ordinarily at least 65%, often at least
68%, more often at least 71%, typically at least 74%, more typically at
least 77%, usually at least 80%, more usually at least about 85%,
preferably at least about 90%, more preferably at least about 95 to 98%
or more, and in particular embodiments, as high at about 99% or more of
the nucleotides. Alternatively, substantial similarity exists when the
segments will hybridize under selective hybridization conditions, to a
strand, or its complement, typically using a sequence derived from SEQ ID
NO: 1, 3, 5 or 7. Typically, selective hybridization will occur when
there is at least about 55% similarity over a stretch of at least about
30 nucleotides, preferably at least about 65% over a stretch of at least
about 25 nucleotides, more preferably at least about 75%, and most
preferably at least about 90% over about 20 nucleotides. See Kanehisa
(1984) Nuc. Acids Res. 12:203-213. The length of similarity comparison,
as described, may be over longer stretches, and in certain embodiments
will be over a stretch of at least about 17 nucleotides, usually at least
about 20 nucleotides, more usually at least about 24 nucleotides,
typically at least about 28 nucleotides, more typically at least about 40
nucleotides, preferably at least about 50 nucleotides, and more
preferably at least about 75 to 100 or more nucleotides, e.g., 150, 200,
etc.

[0040]"Stringent conditions", in referring to homology or substantial
similarity in the hybridization context, will be stringent combined
conditions of salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. The combination of
parameters is more important than the measure of any single parameter.
Stringent temperature conditions will usually include temperatures in
excess of about 30° C., more usually in excess of about 37°
C., typically in excess of about 45° C., more typically in excess
of about 55° C., preferably in excess of about 65° C., and
more preferably in excess of about 70° C. Stringent salt
conditions will ordinarily be less than about 1000 mM, usually less than
about 500 mM, more usually less than about 400 mM, typically less than
about 300 mM, preferably less than about 200 mM, and more preferably less
than about 150 mM. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.
31:349-370. A nucleic acid probe which binds to a target nucleic acid
under stringent conditions is specific for said target nucleic acid. Such
a probe is typically more than 11 nucleotides in length, and is
sufficiently identical or complementary to a target nucleic acid over the
region specified by the sequence of the probe to bind the target under
stringent hybridization conditions.

[0041]CX3Ckines from other mammalian species can be cloned and isolated by
cross-species hybridization of closely related species. See, e.g., below.
Similarity may be relatively low between distantly related species, and
thus hybridization of relatively closely related species is advisable.
Alternatively, preparation of an antibody preparation which exhibits less
species specificity may be useful in expression cloning approaches.

[0042]The phrase "specifically binds to an antibody" or "specifically
immunoreactive with", when referring to a protein or peptide, refers to a
binding reaction which is determinative of the presence of the protein in
the presence of a heterogeneous population of proteins and other
biological components. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein and do not
significantly bind other proteins present in the sample. Specific binding
to an antibody under such conditions may require an antibody that is
selected for its specificity for a particular protein. For example,
antibodies raised to the human CX3Ckine protein immunogen with the amino
acid sequence depicted in SEQ ID NO: 2, 4, 6, or 8 can be selected to
obtain antibodies specifically immunoreactive with CX3Ckine proteins and
not with other proteins. These antibodies recognize proteins highly
similar to the homologous mouse CX3Ckine protein.

III. Nucleic Acids

[0043]Human CX3Ckine is exemplary of a larger class of structurally and
functionally related proteins. These soluble chemokine proteins will
serve to transmit signals between different cell types. The preferred
embodiments, as disclosed, will be useful in standard procedures to
isolate genes from different individuals or other species, e.g., warm
blooded animals, such as birds and mammals. Cross hybridization will
allow isolation of related genes encoding proteins from individuals,
strains, or species. A number of different approaches are available to
successfully isolate a suitable nucleic acid clone based upon the
information provided herein. Southern blot hybridization studies can
qualitatively determine the presence of homologous genes in human,
monkey, rat, dog, cow, and rabbit genomes under specific hybridization
conditions.

[0044]Complementary sequences will also be used as probes or primers.
Based upon identification of the likely amino terminus, other peptides
should be particularly useful, e.g., coupled with anchored vector or
poly-A complementary PCR techniques or with complementary DNA of other
peptides. Moreover, reverse translation using the redundancy in the
genetic code may provide synthetic genes which may encode essentially
identical proteins often with a condo usage selection preferred for
expression in a given host cell.

[0046]There are various methods of isolating DNA sequences encoding
CX3Ckine proteins. For example, DNA is isolated from a genomic or cDNA
library using labeled oligonucleotide probes having sequences identical
or complementary to the sequences disclosed herein. Full-length probes
may be used, or oligonucleotide probes may be generated by comparison of
the sequences disclosed. Such probes can be used directly in
hybridization assays to isolate DNA encoding CX3Ckine proteins, or
primers can be designed, e.g., using flanking sequence, for use in
amplification techniques such as PCR, for the isolation of DNA encoding
CX3Ckine proteins.

[0047]To prepare a cDNA library, mRNA is isolated from cells which express
a CX3Ckine protein. cDNA is prepared from the mRNA and ligated into a
recombinant vector. The vector is transfected into a recombinant host for
propagation, screening, and cloning. Methods for making and screening
cDNA libraries are well known. See Gubler and Hoffman (1983) Gene
25:263-269 and Sambrook, et al.

[0048]For a genomic library, the DNA can be extracted from tissue and
either mechanically sheared or enzymatically digested to yield fragments,
e.g., of about 12-20 kb. The fragments are then separated by gradient
centrifugation and cloned in bacteriophage lambda vectors. These vectors
and phage are packaged in vitro, as described in Sambrook, et al.
Recombinant phage are analyzed by plaque hybridization as described in
Benton and Davis (1977) Science 196:180-182. Colony hybridization is
carried out as generally described in e.g., Grunstein, et al. (1975)
Proc. Natl. Acad. Sci. USA. 72:3961-3965.

[0049]DNA encoding a CX3Ckine protein can be identified in either cDNA or
genomic libraries by its ability to hybridize with the nucleic acid
probes described herein, e.g., in colony or plaque hybridization assays.
The corresponding DNA regions are isolated by standard methods familiar
to those of skill in the art. Seer e.g., Sambrook, et al. Alternatively,
sequence databases, e.g., GenBank, may be evaluated for similar or
corresponding sequences.

[0050]Various methods of amplifying target sequences, such as the
polymerase chain reaction, can also be used to prepare DNA encoding
CX3Ckine proteins. Polymerase chain reaction (PCR) technology is used to
amplify such nucleic acid sequences directly from mRNA, from cDNA, and
from genomic libraries or cDNA libraries. The isolated sequences encoding
CX3Ckine proteins may also be used as templates for PCR amplification.

[0051]Typically, in PCR techniques, oligonucleotide primers complementary
to two 5' regions in two strands of the DNA region to be amplified are
synthesized. The polymerase chain reaction is then carried out using the
two opposite primers. See Innis, et al. (eds.) (1990) PCR Protocols: A
Guide to Methods and Applications Academic Press, San Diego, Calif.
Primers can be selected to amplify the entire regions encoding a
full-length CX3Ckine protein or to amplify smaller DNA segments as
desired. Once such regions are PCR-amplified, they can be sequenced and
oligonucleotide probes can be prepared from sequence obtained using
standard techniques. These probes can then be used to isolate DNA's
encoding CX3Ckine proteins.

[0052]Oligonucleotides for use as probes are usually chemically
synthesized according to the solid phase phosphoramidite triester method
first described by Beaucage and Carruthers (1983) Tetrahedron Lett.
22(20):1859-1862, or using an automated synthesizer, as described in
Needham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.
Purification of oligonucleotides is performed, e.g., by native acrylamide
gel electrophoresis or by anion-exchange HPLC as described in Pearson and
Regnier (1983) J. Chrom. 255:137-149. The sequence of the synthetic
oligonucleotide can be verified using, e.g., the chemical degradation
method of Maxam, A. M. and Gilbert, W. in Grossman, L. and Moldave (eds.)
(1980) Methods in Enzymology 65:499-560 Academic Press, New York.

[0053]An isolated nucleic acid encoding a human CX3Ckine protein was
identified. The nucleotide sequence and corresponding open reading frame
are provided in SEQ ID NO: 1 and 2; with further sequences provided in
SEQ ID NO: 3 and 4. Correspondingly, a mouse sequence was identified and
its nucleotide and corresponding open reading frame are provided as SEQ
ID NO: 5-8.

[0054]These CX3Ckines exhibit limited similarity to portions of
chemokines, particularly the chemokine domains. See, e.g., Matsushima and
Oppenheim (1989) Cytokine 1:2-13; Oppenheim, et al. (1991) Ann. Rev.
Immunol. 9:617-648; Schall (1991) Cytokine 3:165-183, and Gronenborn and
Clore (1991) Protein Engineering 4:263-269. In particular, the human
CX3Ckine shows similarity to the C class of chemokines in the
carboxyl-terminal portion, particularly with respect to length, and at
the positions corresponding, in the numbering of mature human sequence,
to the cys-ala-asp-pro sequence at positions 50-53; and the trp-val at
positions 57-58. CX3Ckines have a much longer carboxyl terminal tail than
the members of the CC or CXC chemokine families, and this "stalk" region
may play a role in chemokine presentation. Notably, the spacing of
conserved cysteine residues in the CXC and CC families of chemokines are
absent in the human CX3Ckine embodiment. Other features of comparison are
apparent between the CX3Ckine and chemokine families. See, e.g., Lodi, et
al. (1994) Science 263:1762-1766. In particular, β-sheet and
α-helix residues can be determined using, e.g., RASMOL program, see
Sayle and Milner-White (1995) TIBS 20:374-376; or Gronenberg, et al.
(1991) Protein Engineering 4:263-269; and other structural features are
defined in Lodi, et al. (1994) Science 263:1762-1767. These secondary and
tertiary features assist in defining further the C, CC, and CXC
structural features, along with spacing of appropriate cysteine residues.

[0055]Based upon the structural modeling and insights in the binding
regions of the chemokines, it is predicted that residues in the mature
human protein, lacking a signal of 24 residues, 26 (his), 28 (gln), 40
(ile), 42 (glu), 47 (arg) and 48 (leu) should be important for chemokine
binding to cells. Residues at the amino terminus are probably not
involved in receptor binding or specificity.

[0056]Moreover, exon boundaries are predicted to correspond to protein
segments including the signal sequence through about the second residue
(his) in the mature protein; from there to about three residues past the
third cys (around the arg-ala); and from there to the end. The third exon
appears to exhibit relatively high similarity to the other chemokines.
The second exon would probably be most characteristic of the CX3C
chemokines, and would be the preferred segment to use to search for
homology in other variants, e.g, species or otherwise. In particular,
segments expected to be preferred in producing CX3C chemokine specific
antibodies will include peptides or sequence in the region from the
second residue of the mature protein (his) through about the third
residue after the third cysteine (arg). Fragments of at least about 8-10
residues in that region would be especially interesting peptides, e.g.,
starting at residue positions of the mature 1, 2, 3, etc. Those fragments
would typically end in that region, e.g., at residue 37, 36, 35, etc.
Other interesting peptides of various lengths would include ones which
begin or end in other positions of the protein, e.g., at residues 87, 86,
etc., with lengths ranging, e.g., from about 8 to 20, 25, 30, 35, 40,
etc. Corresponding fragments of other mammalian CX3Ckine, e.g., mouse,
will be preferred embodiments.

[0057]This invention provides isolated DNA or fragments to encode a
CX3Ckine protein. In addition, this invention provides isolated or
recombinant DNA which encodes a protein or polypeptide which is capable
of hybridizing under appropriate conditions, e.g., high stringency, with
the DNA sequences described herein. Said biologically active protein or
polypeptide can be an intact ligand, or fragment, and have an amino acid
sequence as disclosed in SEQ ID NO: 2, 4, 6, or B. Preferred embodiments
will be full length natural sequences, from isolates, e.g., about 11,000
to 12,500 daltons in size when unglycosylated, or fragments of at least
about 6,000 daltons, more preferably at least about 8,000 daltons. In
glycosylated form, the protein may exceed 12,500 daltons. Further, this
invention contemplates the use of isolated or recombinant DNA, or
fragments thereof, which encode proteins which are homologous to a
CX3Ckine protein or which were isolated using cDNA encoding a CX3Ckine
protein as a probe. The isolated DNA can have the respective regulatory
sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A
addition signals, and others.

IV. Making CX3Ckines

[0058]DNAs which encode a CX3Ckine or fragments thereof can be obtained by
chemical synthesis, screening cDNA libraries, or by screening genomic
libraries prepared from a wide variety of cell lines or tissue samples.
The redundancy of the genetic code provides a number of polynucleotide
sequences which should encode the same protein.

[0059]These DNAs can be expressed in a wide variety of host cells for the
synthesis of a full-length protein or fragments which can in turn, e.g.,
be used to generate polyclonal or monoclonal antibodies; for binding
studies; for construction and expression of modified molecules; and for
structure/function studies. Each CX3Ckine or its fragments, e.g., the
chemokine domain, can be expressed in host cells that are transformed or
transfected with appropriate expression vectors. These molecules can be
substantially purified to be free of protein or cellular contaminants,
other than those derived from the recombinant host, and therefore are
particularly useful in pharmaceutical compositions when combined with a
pharmaceutically acceptable carrier and/or diluent. The antigen, e.g.,
CX3Ckine, or portions thereof, may be expressed as fusions with other
proteins or possessing an epitope tag. Such is applicable also to antigen
binding sites.

[0060]Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired antigen gene or its fragments, usually
operably linked to appropriate genetic control elements that are
recognized in a suitable host cell. The specific type of control elements
necessary to effect expression will depend upon the eventual host cell
used. Generally, the genetic control elements can include a prokaryotic
promoter system or a eukaryotic promoter expression control system, and
typically include a transcriptional promoter, an optional operator to
control the onset of transcription, transcription enhancers to elevate
the level of mRNA expression, a sequence that encodes a suitable ribosome
binding site, and sequences that terminate transcription and translation.
Expression vectors also usually contain an origin of replication that
allows the vector to replicate independently from the host cell.

[0061]The vectors of this invention encompass DNAs which encode a
CX3Ckine, or a fragment thereof, typically encoding, e.g., a biologically
active polypeptide, or protein. The DNA can be under the control of a
viral promoter and can encode a selection marker. This invention further
contemplates use of such expression vectors which are capable of
expressing eukaryotic cDNA coding for a CX3Ckine protein in a prokaryotic
or eukaryotic host, where the vector is compatible with the host and
where the eukaryotic cDNA coding for the protein is inserted into the
vector such that growth of the host containing the vector expresses the
cDNA in question. Usually, expression vectors are designed for stable
replication in their host cells or for amplification to greatly increase
the total number of copies of the desirable gene per cell. It is not
always necessary to require that an expression vector replicate in a host
cell, e.g., it is possible to effect transient expression of the protein
or its fragments in various hosts using vectors that do not contain a
replication origin that is recognized by the host cell. It is also
possible to use vectors that cause integration of a CX3Ckine gene or its
fragments into the host DNA by recombination, or to integrate a promoter
which controls expression of an endogenous gene.

[0062]Vectors, as used herein, contemplate plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the host.
Expression vectors are specialized vectors which contain genetic control
elements that effect expression of operably linked genes. Plasmids are
the most commonly used form of vector, but many other forms of vectors
which serve an equivalent function are suitable for use herein. See,
e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A
Laboratory Manual Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988)
Vectors: A Survey of Molecular Cloning Vectors and Their Uses
Buttersworth, Boston, Mass.

[0063]Suitable host cells include prokaryotes, lower eukaryotes, and
higher eukaryotes. Prokaryotes include both gram negative and gram
positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes
include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus
Dictyostelium. Higher eukaryotes include established tissue culture cell
lines from animal cells, both of non-mammalian origin, e.g., insect
cells, and birds, and of mammalian origin, e.g., human, primates, and
rodents.

[0064]Prokaryotic host-vector systems include a wide variety of vectors
for many different species. As used herein, E. coli and its vectors will
be used generically to include equivalent vectors used in other
prokaryotes. A representative vector for amplifying DNA is pBR322 or its
derivatives. Vectors that can be used to express CX3Ckines or CX3Ckine
fragments include, but are not limited to, such vectors as those
containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp
promoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybrid
promoters such as ptac (pDR540). See Brosius, et al. (1988) "Expression
Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in
Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular Cloning
Vectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.

[0065]Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformed
with CX3Ckine sequence containing vectors. For purposes of this
invention, the most common lower eukaryotic host is the baker's yeast,
Saccharomyces cerevisiae. It will be used generically to represent lower
eukaryotes although a number of other strains and species are also
available. Yeast vectors typically consist of a replication origin
(unless of the integrating type), a selection gene, a promoter, DNA
encoding the desired protein or its fragments, and sequences for
translation termination, polyadenylation, and transcription termination.
Suitable expression vectors for yeast include such constitutive promoters
as 3-phosphoglycerate kinase and various other glycolytic enzyme gene
promoters or such inducible promoters as the alcohol dehydrogenase 2
promoter or metallothionine promoter. Suitable vectors include
derivatives of the following types: self-replicating low copy number
(such as the YRp-series), self-replicating high copy number (such as the
YEp-series); integrating types (such as the YIp-series), or
mini-chromosomes (such as the YCp-series).

[0067]It is likely that CX3Ckines need not be glycosylated to elicit
biological responses. However, it will occasionally be desirable to
express a CX3Ckine polypeptide in a system which provides a specific or
defined glycosylation pattern. In this case, the usual pattern will be
that provided naturally by the expression system. However, the pattern
will be modifiable by exposing the polypeptide, e.g., in unglycosylated
form, to appropriate glycosylating proteins introduced into a
heterologous expression system. For example, the CX3Ckine gene may be
co-transformed with one or more genes encoding mammalian or other
glycosylating enzymes. It is further understood that over glycosylation
may be detrimental to CX3Ckine biological activity, and that one of skill
may perform routine testing to optimize the degree of glycosylation which
confers optimal biological activity.

[0069]Now that CX3Ckines have been characterized, fragments or derivatives
thereof can be prepared by conventional processes for synthesizing
peptides. These include processes such as are described in Stewart and
Young (1984) Solid Phase Peptide Synthesis Pierce Chemical Co., Rockford,
Ill.; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis
Springer-Verlag, New York, N.Y.; and Bodanszky (1984) The Principles of
Peptide Synthesis Springer-Verlag, New York, N.Y. For example, an azide
process, an acid chloride process, an acid anhydride process, a mixed
anhydride process, an active ester process (for example, p-nitrophenyl
ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a
carbodiimidazole process, an oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid phase
and solution phase syntheses are both applicable to the foregoing
processes.

[0070]The prepared protein and fragments thereof can be isolated and
purified from the reaction mixture by means of peptide separation, for
example, by extraction, precipitation, electrophoresis and various forms
of chromatography, and the like. The CX3Ckines of this invention can be
obtained in varying degrees of purity depending upon its desired use.
Purification can be accomplished by use of known protein purification
techniques or by the use of the antibodies or binding partners herein
described, e.g., in immunoabsorbant affinity chromatography. See, e.g.,
Coilgan, et al. (eds.) (1995 and periodic supplements) Current Protocols
in Protein Science, John Wiley and Sons, New York, N.Y. This
immunoabsorbant affinity chromatography is carried out by first linking
the antibodies to a solid support and then contacting the linked
antibodies with solubilized lysates of appropriate source cells, lysates
of other cells expressing the ligand, or lysates or supernatants of cells
producing the CX3Ckines as a result of recombinant DNA techniques, see
below.

[0071]Multiple cell lines may be screened for one which expresses a
CX3Ckine at a high level compared with other cells. Various cell lines,
e.g., a mouse thymic stromal cell line TA4, is screened and selected for
its favorable handling properties. Natural CX3Ckines can be isolated from
natural sources, or by expression from a transformed cell using an
appropriate expression vector. Purification of the expressed protein is
achieved by standard procedures, or may be combined with engineered means
for effective purification at high efficiency from cell lysates or
supernatants. Epitope or other tags, e.g., FLAG or His6 segments,
can be used for such purification features.

V. Antibodies

[0072]Antibodies can be raised to various CX3Ckines, including individual,
polymorphic, allelic, strain, or species variants, and fragments thereof,
both in their naturally occurring (full-length) forms and in their
recombinant forms. Additionally, antibodies can be raised to CX3Ckines in
either their active or native forms or in their inactive or denatured
forms. Anti-idiotypic antibodies may also be used.

[0073]A. Antibody Production

[0074]A number of immunogens may be used to produce antibodies
specifically reactive with CX3Ckine proteins. Recombinant protein is a
preferred immunogen for the production of monoclonal or polyclonal
antibodies. Naturally occurring protein may also be used either in pure
or impure form. Synthetic peptides, made using the human or mouse
CX3Ckine protein sequences described herein, may also used as an
immunogen for the production of antibodies to CX3Ckines, e.g., the
chemokine domains thereof. Recombinant protein can be expressed in
eukaryotic or prokaryotic cells as described herein, and purified as
described. Naturally folded or denatured material can be used, as
appropriate, for producing antibodies. Either monoclonal or polyclonal
antibodies may be generated for subsequent use in immunoassays to measure
the protein.

[0075]Methods of producing polyclonal antibodies are known to those of
skill in the art. Typically, an immunogen, preferably a purified protein,
is mixed with an adjuvant and animals are immunized with the mixture. The
animal's immune response to the immunogen preparation is monitored by
taking test bleeds and determining the titer of reactivity to the
CX3Ckine protein or fragment of interest. When appropriately high titers
of antibody to the immunogen are obtained, usually after repeated
immunizations, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for antibodies
reactive to the protein can be done if desired. See, e.g., Harlow and
Lane; or Coligan.

[0076]Monoclonal antibodies may be obtained by various techniques familiar
to those skilled in the art. Typically, spleen cells from an animal
immunized with a desired antigen are immortalized, commonly by fusion
with a myeloma cell (see, Kohler and Milstein (1976) Eur. J. Immunol.
6:511-519, incorporated herein by reference). Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. Colonies
arising from single immortalized cells are screened for production of
antibodies of the desired specificity and affinity for the antigen, and
yield of the monoclonal antibodies produced by such cells may be enhanced
by various techniques, including injection into the peritoneal cavity of
a vertebrate host. Alternatively, one may isolate DNA sequences which
encode a monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according, e.g., to the general protocol
outlined by Huse, et al. (1989) Science 246:1275-1281.

[0077]Antibodies, including binding fragments and single chain versions,
against predetermined fragments of CX3Ckines can be raised by
immunization of animals with conjugates of the fragments with carrier
proteins as described above. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be screened
for binding to normal or defective CX3Ckines, or screened for agonistic
or antagonistic activity, e.g., mediated through a receptor. These
monoclonal antibodies will usually bind with at least a KD of about
1 mM, more usually at least about 300 μM, typically at least about 10
μM, more typically at least about 30 μM, preferably at least about
10 μM, and more preferably at least about 3 μM or better.

[0078]In some instances, it is desirable to prepare monoclonal antibodies
from various mammalian hosts, such as mice, rodents, primates, humans,
etc. Description of techniques for preparing such monoclonal antibodies
may be found in, e.g., Stites, et al. (eds.) Basic and Clinical
Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and
references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory
Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and
Practice (2d ed.) Academic Press, New York, N.Y.; and particularly in
Kohler and Milstein (1975) Nature 256:495-497, which discusses one method
of generating monoclonal antibodies. Summarized briefly, this method
involves injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused with
myeloma cells. The result is a hybrid cell or "hybridoma" that is capable
of reproducing in vitro. The population of hybridomas is then screened to
isolate individual clones, each of which secrete a single antibody
species to the immunogen. In this manner, the individual antibody species
obtained are the products of immortalized and cloned single B cells from
the immune animal generated in response to a specific site recognized on
the immunogenic substance.

[0079]Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse, et al. (1989)
"Generation of a Large Combinatorial Library of the Immunoglobulin
Repertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al.
(1989) Nature 341:544-546. The polypeptides and antibodies of the present
invention may be used with or without modification, including chimeric or
humanized antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a substance
which provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in both the
scientific and patent literature. Suitable labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent moieties,
chemiluminescent moieties, magnetic particles, and the like. Patents,
teaching the use of such labels include U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S.
Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033.

[0080]The antibodies of this invention are useful for affinity
chromatography in isolating CX3Ckine protein. Columns can be prepared
where the antibodies are linked to a solid support, e.g., particles, such
as agarose, SEPHADEX, or the like, where a cell lysate or supernatant may
be passed through the column, the column washed, followed by increasing
concentrations of a mild denaturant, whereby purified CX3Ckine protein
will be released.

[0081]The antibodies may also be used to screen expression libraries for
particular expression products. Usually the antibodies used in such a
procedure will be labeled with a moiety allowing easy detection of
presence of antigen by antibody binding.

[0082]Antibodies to CX3Ckines may be used for the identification of cell
populations expressing CX3Ckines. By assaying the expression products of
cells expressing CX3Ckines it is possible to diagnose disease, e.g.,
immune-compromised conditions.

[0083]Antibodies raised against each CX3Ckine will also be useful to raise
anti-idiotypic antibodies. These will be useful in detecting or
diagnosing various immunological conditions related to expression of the
respective antigens.

[0084]B. Immunoassays

[0085]A particular protein can be measured by a variety of immunoassay
methods. For a review of immunological and immunoassay procedures in
general, see Stites and Terr (eds.) (1991) Basic and Clinical Immunology
(7th ed.). Moreover, the immunoassays of the present invention can be
performed in many configurations, which are reviewed extensively in
Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan
(1985) "Practice and Theory of Enzyme Immunoassays," Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers B.V., Amsterdam; and Harlow and Lane Antibodies, A Laboratory
Manual, supra, each of which is incorporated herein by reference. See
also Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press,
Orlando, Fla.; Price and Newman (eds.) (1991) Principles and Practice of
Immunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic
Immunoassays Plenum Press, NY.

[0086]Immunoassays for measurement of CX3Ckine proteins can be performed
by a variety of methods known to those skilled in the art. In brief,
immunoassays to measure the protein can be competitive or noncompetitive
binding assays. In competitive binding assays, the sample to be analyzed
competes with a labeled analyte for specific binding sites on a capture
agent bound to a solid surface. Preferably the capture agent is an
antibody specifically reactive with CX3Ckine proteins produced as
described above. The concentration of labeled analyte bound to the
capture agent is inversely proportional to the amount of free analyte
present in the sample.

[0087]In a competitive binding immunoassay, the CX3Ckine protein present
in the sample competes with labeled protein for binding to a specific
binding agent, for example, an antibody specifically reactive with the
CX3Ckine protein. The binding agent may be bound to a solid surface to
effect separation of bound labeled protein from the unbound labeled
protein. Alternatively, the competitive binding assay may be conducted in
liquid phase and a variety of techniques known in the art may be used to
separate the bound labelled protein from the unbound labeled protein.
Following separation, the amount of bound labeled protein is determined.
The amount of protein present in the sample is inversely proportional to
the amount of labeled protein binding.

[0088]Alternatively, a homogeneous immunoassay may be performed in which a
separation step is not needed. In these immunoassays, the label on the
protein is altered by the binding of the protein to its specific binding
agent. This alteration in the labeled protein results in a decrease or
increase in the signal emitted by label, so that measurement of the label
at the end of the immunoassay allows for detection or quantitation of the
protein.

[0089]CX3Ckine proteins may also be determined by a variety of
noncompetitive immunoassay methods. For example, a two-site, solid phase
sandwich immunoassay may be used. In this type of assay, a binding agent
for the protein, for example an antibody, is attached to a solid support.
A second protein binding agent, which may also be an antibody, and which
binds the protein at a different site, is labelled. After binding at both
sites on the protein has occurred, the unbound labeled binding agent is
removed and the amount of labeled binding agent bound to the solid phase
is measured. The amount of labeled binding agent bound is directly
proportional to the amount of protein in the sample.

[0090]Western blot analysis can be used to determine the presence of
CX3Ckine proteins in a sample. Electrophoresis is carried out, for
example, on a tissue sample suspected of containing the protein.
Following electrophoresis to separate the proteins, and transfer of the
proteins to a suitable solid support, e.g., a nitrocellulose filter, the
solid support is incubated with an antibody reactive with the protein.
This antibody may be labeled, or alternatively may be detected by
subsequent incubation with a second labeled antibody that binds the
primary antibody.

[0091]The immunoassay formats described above employ labeled assay
components. The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in the
art. A wide variety of labels and methods may be used. Traditionally, a
radioactive label incorporating 3H, 125I, 35S, 14C,
or 32P was used. Non-radioactive labels include ligands which bind
to labeled antibodies, fluorophores, chemiluminescent agents, enzymes,
and antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity required, ease
of conjugation with the compound, stability requirements, and available
instrumentation. For a review of various labelling or signal producing
systems which may be used, see U.S. Pat. No. 4,391,904, which is
incorporated herein by reference.

[0092]Antibodies reactive with a particular protein can also be measured
by a variety of immunoassay methods. For a review of immunological and
immunoassay procedures applicable to the measurement of antibodies by
immunoassay techniques, see Stites and Terr (eds.) Basic and Clinical
Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; and
Harlow and Lane Antibodies, A Laboratory Manual, supra.

[0093]In brief, immunoassays to measure antisera reactive with CX3Ckine
proteins can be competitive or noncompetitive binding assays. In
competitive binding assays, the sample analyte competes with a labeled
analyte for specific binding sites on a capture agent bound to a solid
surface. Preferably the capture agent is a purified recombinant CX3Ckine
protein produced as described above. Other sources of CX3Ckine proteins,
including isolated or partially purified naturally occurring protein, may
also be used. Noncompetitive assays include sandwich assays, in which the
sample analyte is bound between two analyte-specific binding reagents.
One of the binding agents is used as a capture agent and is bound to a
solid surface. The second binding agent is labeled and is used to measure
or detect the resultant complex by visual or instrument means. A number
of combinations of capture agent and labelled binding agent can be used.
A variety of different immunoassay formats, separation techniques, and
labels can be also be used similar to those described above for the
measurement of CX3Ckine proteins.

[0095]Purified protein or defined peptides are useful for generating
antibodies by standard methods, as described above. Synthetic peptides or
purified protein, e.g., the chemokine domains, can be presented to an
immune system to generate polyclonal and monoclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring
Harbor Press, NY, which are incorporated herein by reference.
Alternatively, a CX3Ckine receptor can be useful as a specific binding
reagent, and advantage can be taken of its specificity of binding, for,
e.g., purification of a CX3Ckine ligand.

[0096]The specific binding composition can be used for screening an
expression library made from a cell line which expresses a CX3Ckine. Many
methods for screening are available, e.g., standard staining of surface
expressed ligand, or by panning. Screening of intracellular expression
can also be performed by various staining or immunofluorescence
procedures. The binding compositions could be used to affinity purify or
sort out cells expressing the ligand.

[0097]The peptide segments, along with comparison to homologous genes, can
also be used to produce appropriate oligonucleotides to screen a library.
The genetic code can be used to select appropriate oligonucleotides
useful as probes for screening. In combination with polymerase chain
reaction (PCR) techniques, synthetic oligonucleotides will be useful in
selecting desired clones from a library, including natural allelic an
polymorphic variants.

[0098]The peptide sequences allow preparation of peptides to generate
antibodies to recognize such segments, and allow preparation of
oligonucleotides which encode such sequences. The sequence also allows
for synthetic preparation, e.g., see Dawson, et al. (1994) Science
266:776-779. Since CX3Ckines appear to be soluble proteins, the gene will
normally possess an N-terminal signal sequence, which is removed upon
processing and secretion, and the putative cleavage site is between amino
acids 24 (gly) and 25 (gln) in SEQ ID NO: 2 or 4, though it may be
slightly in either direction. Analysis of the structural features in
comparison with the most closely related reported sequences has revealed
similarities with other cytokines, particularly the class of proteins
known as chemokines. Within the chemokines are two subgroups, the CC and
CXC subgroups. The CX3Ckine family shares various features with each of
these groups, but its combination of features is distinctive and defines
a new family of related chemokines.

[0099]While further structural features result from the sequences provided
in SEQ ID NO: 1 through 8, the "chemokine on a stick" feature is provided
through the stalk region which possesses many sites which may provide a
heavily glycosylated domain. The stalk structure may be important in CX3C
chemokine presentation to other cells. In fact, it appears that the stalk
region may be processed to release the soluble chemokine. This suggests
the possibility of substituting the CX3C chemokine domain with other
chemokines to effect efficient presentation to appropriate target cells.

[0100]In addition, the "stalk" regions are likely to affect solubility and
pharmacological aspects of the protein. As such, this region will be the
target of analysis to evaluate and modulate such features as
pharmacokenetics. Truncation of that portion may affect half-life,
clearance, and accessibility of the chemokine domains.

VII. Physical Variants

[0101]This invention also encompasses proteins or peptides having
substantial amino acid sequence similarity with an amino acid sequence of
a CX3Ckine. Natural variants include individual, polymorphic, allelic,
strain, or species variants.

[0102]Amino acid sequence similarity, or sequence identity, is determined
by optimizing residue matches, if necessary, by introducing gaps as
required. This changes when considering conservative substitutions as
matches. Conservative substitutions typically include substitutions
within the following groups: glycine, alanine; valine, isoleucine,
leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,
threonine; lysine, arginine; and phenylalanine, tyrosine. Homologous
amino acid sequences include natural polymorphic, allelic, and
interspecies variations in each respective protein sequence. Typical
homologous proteins or peptides will have from 50-100% similarity (if
gaps can be introduced), to 75-100% similarity (if conservative
substitutions are included) with the amino acid sequence of the CX3Ckine.
Similarity measures will be at least about 50%, generally at least 60%,
more generally at least 65%, usually at least 70%, more usually at least
75%, preferably at least 80%, and more preferably at least 80%, and in
particularly preferred embodiments, at least 85% or more. See also
Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983)
Time Warps, String Edits, and Macromolecules: The Theory and Practice of
Sequence Comparison Chapter One, Addison-Wesley, Reading, Mass.; and
software packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.

[0103]Nucleic acids encoding mammalian CX3Ckine proteins will typically
hybridize to the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 under
stringent conditions. For example, nucleic acids encoding human CX3Ckine
proteins will normally hybridize to the nucleic acid of SEQ ID NO: 1
under stringent hybridization conditions. Generally, stringent conditions
are selected to be about 10° C. lower than the thermal melting
point (Tm) for the probe sequence at a defined ionic strength and pH. The
Tm is the temperature (under defined ionic strength and pH) at which 50%
of the target sequence hybridizes to a perfectly matched probe.
Typically, stringent conditions will be those in which the salt
concentration is about 0.2 molar at pH 7 and the temperature is at least
about 50° C. Other factors may significantly affect the stringency
of hybridization, including, among others, base composition and size of
the complementary strands, the presence of organic solvents such as
formamide, and the extent of base mismatching. A preferred embodiment
will include nucleic acids which will bind to disclosed sequences in 50%
formamide and 200 mM NaCl at 42° C.

[0104]An isolated CX3Ckine DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and short
inversions of nucleotide stretches. These modifications result in novel
DNA sequences which encode CX3Ckine antigens, their derivatives, or
proteins having highly similar physiological, immunogenic, or antigenic
activity.

[0105]Modified sequences can be used to produce mutant antigens or to
enhance expression. Enhanced expression may involve gene amplification,
increased transcription, increased translation, and other mechanisms.
Such mutant CX3Ckine derivatives include predetermined or site-specific
mutations of the respective protein or its fragments. "Mutant CX3Ckine"
encompasses a polypeptide otherwise falling within the homology
definition of the human CX3Ckine as set forth above, but having an amino
acid sequence which differs from that of a CX3Ckine as found in nature,
whether by way of deletion, substitution, or insertion. In particular,
"site specific mutant CX3Ckine" generally includes proteins having
significant similarity with a protein having a sequence of SEQ ID NO: 2,
4, 6, or 8, and as sharing various biological activities, e.g., antigenic
or immunogenic, with those sequences, and in preferred embodiments
contain most or all of the disclosed sequence. This applies also to
polymorphic variants from different individuals. Similar concepts apply
to different CX3Ckine proteins, particularly those found in various warm
blooded animals, e.g., mammals and birds. As stated before, it is
emphasized that descriptions are generally meant to encompass other
CX3Ckine proteins, not limited to the human or mouse embodiments
specifically discussed.

[0106]Although site specific mutation sites are predetermined, mutants
need not be site specific. CX3Ckine mutagenesis can be conducted by
making amino acid insertions or deletions. Substitutions, deletions,
insertions, or any combinations may be generated to arrive at a final
construct. These include amino acid residue substitution levels from
none, one, two, three, five, seven, ten, twelve, fifteen, etc. Insertions
include amino- or carboxyl-terminal fusions, e.g. epitope tags. Random
mutagenesis can be conducted at a target codon and the expressed mutants
can then be screened for the desired activity. Methods for making
substitution mutations at predetermined sites in DNA having a known
sequence are well known in the art, e.g., by M13 primer mutagenesis or
polymerase chain reaction (PCR) techniques. See also, Sambrook, et al.
(1989) and Ausubel, et al. (1987 and Supplements). The mutations in the
DNA normally should not place coding sequences out of reading frames and
preferably will not create complementary regions that could hybridize to
produce secondary mRNA structure such as loops or hairpins.

[0107]The present invention also provides recombinant proteins, e.g.,
heterologous fusion proteins using segments from these proteins, both the
CX3Ckine, or antigen binding sites. A heterologous fusion protein is a
fusion of proteins or segments which are naturally not normally fused in
the same manner. Thus, the fusion product of an immunoglobulin with a
CX3Ckine polypeptide is a continuous protein molecule having sequences
fused in a typical peptide linkage, typically made as a single
translation product and exhibiting properties derived from each source
peptide. A similar concept applies to heterologous nucleic acid
sequences.

[0108]In addition, new constructs may be made from combining similar
functional domains from other proteins. For example, protein-binding or
other segments may be "swapped" between different new fusion polypeptides
or fragments. See, e.g., Cunningham, et al. (1989) Science 243:1330-1336;
and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992. Thus, new
chimeric polypeptides exhibiting new combinations of specificities will
result from the functional linkage of protein-binding specificities and
other functional domains.

VIII. Binding Agent:CX3Ckine Protein Complexes

[0109]A CX3Ckine protein that specifically binds to or that is
specifically immunoreactive with an antibody generated against a defined
immunogen, such as an immunogen consisting of the amino acid sequence of
SEQ ID NO: 2. 4, 6, or 8, is typically determined in an immunoassay. The
immunoassay uses a polyclonal antiserum which was raised to a protein of
SEQ ID NO: 2. 4, 6, or 8. This antiserum is selected to have low
crossreactivity against other chemokines and any such crossreactivity is
removed by immunoabsorbtion prior to use in the immunoassay.

[0110]In order to produce antisera for use in an immunoassay, the protein
of SEQ ID NO: 2. 4, 6, or 3, is isolated as described herein. For
example, recombinant protein may be produced in a mammalian cell line. An
inbred strain of mice such as balb/c is immunized with the protein of SEQ
ID NO: 2. 4, 6, or 8, using a standard adjuvant, such as Freund's
adjuvant, and a standard mouse immunization protocol (see Harlow and
Lane, supra). Alternatively, a synthetic peptide, preferably near full
length, derived from the sequences disclosed herein and conjugated to a
carrier protein can be used an immunogen. Polyclonal sera are collected
and titered against the immunogen protein in an immunoassay, for example,
a solid phase immunoassay with the immunogen immobilized on a solid
support. Polyclonal antisera with a titer of 104 or greater are selected
and tested for their cross reactivity against C, C--C, and CXC
chemokines, using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573. Preferably two
chemokines are used in this determination in conjunction with either
human CX3Ckine or mouse CX3Ckine.

[0111]In conjunction with a CX3Ckine, the monocyte chemotactic protein-1
(MCP-1) and macrophage inflammatory protein-1α (Mip-1α) are
used to identify antibodies which are specifically bound by a CX3Ckine.
In conjunction with human CX3Ckine, the monocyte chemotactic protein-2
(MCP-2) and Mip-1α are used to identify antibodies which are
specifically bound by a CX3Ckine. These chemokines can be produced as
recombinant proteins and isolated using standard molecular biology and
protein chemistry techniques as described herein.

[0112]Immunoassays in the competitive binding format can be used for the
crossreactivity determinations. For example, a protein of SEQ ID NO: 2.
4, 6, or 8 can be immobilized to a solid support. Proteins added to the
assay compete with the binding of the antisera to the immobilized
antigen. The ability of the above proteins to compete with the binding of
the antisera to the immobilized protein is compared to the protein of SEQ
ID NO: 2. 4, 6, or 8. The percent crossreactivity for the above proteins
is calculated, using standard calculations. Those antisera with less than
10% crossreactivity with each of the proteins listed above are selected
and pooled. The cross-reacting antibodies are then removed from the
pooled antisera by immunoabsorbtion with the above-listed proteins.

[0113]The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a second
protein to the immunogen protein (e.g., the CX3Ckine chemokine motif of
SEQ ID NO: 2. 4, 6, or 8). In order to make this comparison, the two
proteins are each assayed at a wide range of concentrations and the
amount of each protein required to inhibit 50% of the binding of the
antisera to the immobilized protein is determined. If the amount of the
second protein required is less than twice the amount of the protein of
SEQ ID NO: 2 that is required, then the second protein is said to
specifically bind to an antibody generated to the immunogen.

[0114]It is understood that CX3Ckine proteins are a family of homologous
proteins that comprise two or more genes. For a particular gene product,
such as the human CX3Ckine protein, the term refers not only to the amino
acid sequences disclosed herein, but also to other proteins that are
polymorphic, allelic, non-allelic, or species variants. It is also
understood that the term "human CX3Ckine" or "mouse CX3Ckine" includes
nonnatural mutations introduced by deliberate mutation using conventional
recombinant technology such as single site mutation, or by excising short
sections of DNA encoding CX3Ckine proteins, or by substituting new amino
acids, or adding new amino acids. Such minor alterations must
substantially maintain the immunoidentity of the original molecule and/or
its biological activity. Thus, these alterations include proteins that
are specifically immunoreactive with a designated naturally occurring
CX3Ckine protein, for example, the human CX3Ckine protein shown in SEQ ID
NO: 2 or 4. The biological properties of the altered proteins can be
determined by expressing the protein in an appropriate cell line and
measuring, e.g., a chemotactic effect. Particular protein modifications
considered minor would include conservative substitution of amino acids
with similar chemical properties, as described above for the CX3Ckine
family as a whole. By aligning a protein optimally with the protein of
SEQ ID NO: 2. 4, 6, or 8, and by using the conventional immunoassays
described herein to determine immunoidentity, or by using lymphocyte
chemotaxis assays, one can determine the protein compositions of the
invention.

IX. Functional Variants

[0115]The blocking of physiological response to CX3Ckines may result from
the inhibition of binding of the protein to its receptor, e.g., through
competitive inhibition. Thus, in vitro assays of the present invention
will often use isolated protein, membranes from cells expressing a
recombinant membrane associated CX3Ckine, soluble fragments comprising
receptor binding segments of these proteins, or fragments attached to
solid phase substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations and
modifications, or protein mutations and modifications, e.g., protein
analogs. This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to antigen or
receptor fragments compete with a test compound for binding to the
protein. In this manner, the antibodies can be used to detect the
presence of a polypeptide which shares one or more antigenic binding
sites of the protein and can also be used to occupy binding sites on the
protein that might otherwise interact with a receptor.

[0116]"Derivatives" of CX3Ckine antigens include amino acid sequence
mutants, glycosylation variants, and covalent or aggregate conjugates
with other chemical moieties. Covalent derivatives can be prepared by
linkage of functionalities to groups which are found in CX3Ckine amino
acid side chains or at the N- or C-termini, by means which are well known
in the art. These derivatives can include, without limitation, aliphatic
esters or amides of the carboxyl terminus, or of residues containing
carboxyl side chains, O-acyl derivatives of hydroxyl group-containing
residues, and N-acyl derivatives of the amino terminal amino acid or
amino-group containing residues, e.g., lysine or arginine. Acyl groups
are selected from the group of alkyl-moieties including C3 to C18 normal
alkyl, thereby forming alkanoyl aroyl species. See, e.g., Coligan, et al.
(eds.) (1995 and periodic supplements) Current Protocols in Protein
Science, John Wiley and Sons, New York, N.Y. Covalent attachment to
carrier proteins may be important when immunogenic moieties are haptens.

[0117]In particular, glycosylation alterations are included, e.g., made by
modifying the glycosylation patterns of a polypeptide during its
synthesis and processing, or in further processing steps. Particularly
preferred means for accomplishing this are by exposing the polypeptide to
glycosylating enzymes derived from cells which normally provide such
processing, e.g., a mammalian glycosylation enzymes. Deglycosylation
enzymes are also contemplated. Also embraced are versions of the same
primary amino acid sequence which have other minor modifications,
including phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine, or other moieties, including ribosyl
groups or cross-linking reagents.

[0118]A major group of derivatives are covalent conjugates of the CX3Ckine
or fragments thereof with other proteins or polypeptides. These
derivatives can be synthesized in recombinant culture such as N- or
C-terminal fusions or by the use of agents known in the art for their
usefulness in cross-linking proteins through reactive side groups.
Preferred protein derivatization sites with cross-linking agents are at
free amino groups, carbohydrate moieties, and cysteine residues.

[0119]Fusion polypeptides between CX3Ckines and other homologous or
heterologous proteins are also provided. Many growth factors and
cytokines are homodimeric entities, and a repeat construct may have
various advantages, including lessened susceptibility to proteolytic
degradation. Moreover, many receptors require dimerization to transduce a
signal, and various dimeric proteins or domain repeats can be desirable.
Heterologous polypeptides may be fusions between different surface
markers, resulting in, e.g., a hybrid protein exhibiting receptor binding
specificity. Likewise, heterologous fusions may be constructed which
would exhibit a combination of properties or activities of the derivative
proteins. Typical examples are fusions of a reporter polypeptide, e.g.,
luciferase, with a segment or domain of a protein, e.g., a
receptor-binding segment, so that the presence or location of the fused
protein may be easily determined. See, e.g., Dull, et al., U.S. Pat. No.
4,859,609. Other gene fusion partners include bacterial
β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,
alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., See,
e.g., Dawson, et al. (1994) Science 266:776-779; and Godowski, et al.
(1988) Science 241:812-816. In particular, fusion proteins with portions
from the related genes will be useful. Similar concepts of fusions with
antigen binding sites are contemplated.

[0120]Such polypeptides may also have amino acid residues which have been
chemically modified by phosphorylation, sulfonation, biotinylation, or
the addition or removal of other moieties, particularly those which have
molecular shapes similar to phosphate groups. In some embodiments, the
modifications will be useful labeling reagents, or serve as purification
targets, e.g., affinity ligands.

[0121]This invention also contemplates the use of derivatives of CX3Ckines
other than variations in amino acid sequence or glycosylation. Such
derivatives may involve covalent or aggregative association with chemical
moieties. These derivatives include: (1) salts, (2) side chain and
terminal residue covalent modifications, and (3) adsorption complexes,
for example with cell membranes. Such covalent or aggregative derivatives
are useful as immunogens, as reagents in immunoassays, or in purification
methods such as for affinity purification of ligands or other binding
ligands. For example, a CX3Ckine antigen can be immobilized by covalent
bonding to a solid support such as cyanogen bromide-activated SEPHAROSE,
by methods which are well known in the art, or adsorbed onto polyolefin
surfaces, with or without glutaraldehyde cross-linking, for use in the
assay or purification of anti-CX3Ckine antibodies or its receptor. The
CX3Ckines can also be labeled with a detectable group, e.g.,
radioiodinated by the chloramine T procedure, covalently bound to rare
earth chelates, or conjugated to another fluorescent moiety for use in
diagnostic assays. Purification of CX3Ckines may be effected by
immobilized antibodies or receptor.

[0122]Isolated CX3Ckine genes will allow transformation of cells lacking
expression of corresponding CX3Ckines, e.g., either species types or
cells which Lack corresponding proteins and exhibit negative background
activity. Expression of transformed genes will allow isolation of
antigenically pure cell lines, with defined or single specie variants.
This approach will allow for more sensitive detection and discrimination
of the physiological effects of CX3Ckine receptor proteins. Subcellular
fragments, e.g., cytoplasts or membrane fragments, can be isolated and
used.

X. Uses

[0123]The present invention provides reagents which will find use in
diagnostic applications as described elsewhere herein, e.g., in the
general description for developmental abnormalities, or below in the
description of kits for diagnosis.

[0124]CX3Ckine nucleotides, e.g., human or mouse CX3Ckine DNA or RNA, may
be used as a component in a forensic assay. For instance, the nucleotide
sequences provided may be labeled using, e.g., 32P or biotin and
used to probe standard restriction fragment polymorphism blots, providing
a measurable character to aid in distinguishing between individuals. Such
probes may be used in well-known forensic techniques such as genetic
fingerprinting. In addition, nucleotide probes made from CX3Ckine
sequences may be used in in situ assays to detect chromosomal
abnormalities. For instance, rearrangements in the mouse chromosome
encoding a CX3Ckine gene may be detected via well-known in situ
techniques, using CX3Ckine probes in conjunction with other known
chromosome markers.

[0125]Antibodies and other binding agents directed towards CX3Ckine
proteins or nucleic acids may be used to purify the corresponding
CX3Ckine molecule. As described in the Examples below, antibody
purification of CX3Ckine components is both possible and practicable.
Antibodies and other binding agents may also be used in a diagnostic
fashion to determine whether CX3Ckine components are present in a tissue
sample or cell population using well-known techniques described herein.
The ability to attach a binding agent to a CX3Ckine provides a means to
diagnose disorders associated with CX3Ckine misregulation. Antibodies and
other CX3Ckine binding agents may also be useful as histological markers.
As described in the examples below, CX3Ckine expression is limited to
specific tissue types. By directing a probe, such as an antibody or
nucleic acid to a CX3Ckine it is possible to use the probe to distinguish
tissue and cell types in situ or in vitro.

[0126]This invention also provides reagents with significant therapeutic
value. The CX3Ckines (naturally occurring or recombinant), fragments
thereof, and antibodies thereto, along with compounds identified as
having binding affinity to a CX3Ckine, are useful in the treatment of
conditions associated with abnormal physiology or development, including
abnormal proliferation, e.g., cancerous conditions, or degenerative
conditions. Abnormal proliferation, regeneration, degeneration, and
atrophy may be modulated by appropriate therapeutic treatment using the
compositions provided herein. For example, a disease or disorder
associated with abnormal expression or abnormal signaling by a CX3Ckine
is a target for an agonist or antagonist of the protein. The proteins
likely play a role in regulation or development of neuronal or
hematopoietic cells, e.g., lymphoid cells, which affect immunological
responses.

[0127]Other abnormal developmental conditions are known in cell types
shown to possess CX3Ckine mRNA by northern blot analysis. See Berkow
(ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,
N.J.; and Thorn, et al. Harrison's Principles of Internal Medicine,
McGraw-Hill, NY. Developmental or functional abnormalities, e.g., of the
neuronal or immune system, cause significant medical abnormalities and
conditions which may be susceptible to prevention or treatment using
compositions provided herein.

[0128]Certain chemokines have also been implicated in viral replication
mechanisms. See, e.g., Cohen (1996) Science 272:809-810; Feng, et al.
(1996) Science 272:872-877; and Cocchi, et al. (1995) Science
270:1811-1816. The CX3C chemokine may be useful in a similar context.
Alternatively, the stalk structure may be very important in presentation
of the ligand domain, and other chemokines may be advantageously
substituted for the chemokine domain in this molecule. Modification in
the "stalk" structure may affect many of the pharmacological properties
of the CX3Ckine, including half-life and biological activity.

[0129]Recombinant CX3Ckine or CX3Ckine antibodies can be purified and then
administered to a patient, e.g., in sterile form. These reagents can be
combined for therapeutic use with additional active or inert ingredients,
e.g., in conventional pharmaceutically acceptable carriers or diluents,
e.g., immunogenic adjuvants, along with physiologically innocuous
stabilizers and excipients. These combinations can be sterile filtered
and placed into dosage forms as by lyophilization in dosage vials or
storage in stabilized aqueous preparations. This invention also
contemplates use of antibodies or binding fragments thereof, including
forms which are not complement binding.

[0130]Drug screening using antibodies or receptor or fragments thereof can
identify compounds having binding affinity to CX3Ckines, including
isolation of associated components. Subsequent biological assays can then
be utilized to determine if the compound has intrinsic stimulating
activity and is therefore a blocker or antagonist in that it blocks the
activity of the protein. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is thus an agonist in
that it simulates the activity of a CX3Ckine. This invention further
contemplates the therapeutic use of antibodies to CX3Ckines as
antagonists. This approach should be particularly useful with other
CX3Ckine species variants.

[0131]The quantities of reagents necessary for effective therapy will
depend upon many different factors, including means of administration,
target site, physiological state of the patient, and other medicants
administered. Thus, treatment dosages should be titrated to optimize
safety and efficacy. Typically, dosages used in vitro may provide useful
guidance in the amounts useful for in situ administration of these
reagents. Animal testing of effective doses for treatment of particular
disorders will provide further predictive indication of human dosage.
Various considerations are described, e.g., in Gilman, et al. (eds.)
(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics
(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences
(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administration
are discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal diffusion,
and others. Pharmaceutically acceptable carriers will include water,
saline, buffers, and other compounds described, e.g., in the Merck Index,
Merck & Co., Rahway, N.J. Dosage ranges would ordinarily be expected to
be in amounts lower than 1 mM concentrations, typically less than about
10 μM concentrations, usually less than about 100 nM, preferably less
than about 10 μM (picomolar), and most preferably less than about 1 fM
(femtomolar), with an appropriate carrier. Slow release formulations, or
a slow release apparatus will often be utilized for continuous
administration.

[0132]CX3Ckines, fragments thereof, and antibodies to it or its fragments,
antagonists, and agonists, may be administered directly to the host to be
treated or, depending on the size of the compounds, it may be desirable
to conjugate them to carrier proteins such as ovalbumin or serum albumin
prior to their administration. Therapeutic formulations may be
administered in many conventional dosage formulations. While it is
possible for the active ingredient to be administered alone, it is
preferable to present it as a pharmaceutical formulation. Formulations
typically comprise at least one active ingredient, as defined above,
together with one or more acceptable carriers thereof. Each carrier
should be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not injurious to
the patient. Formulations include those suitable for oral, rectal, nasal,
or parenteral (including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods well
known in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th ed.)
Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th ed.)
Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, N.Y.;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosgae Forms: Tablets
Dekker, N.Y.; and Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage
Forms: Disperse Systems Dekker, N.Y. The therapy of this invention may be
combined with or used in association with other therapeutic agents.

[0133]Both the naturally occurring and the recombinant forms of the
CX3Ckines of this invention are particularly useful in kits and assay
methods which are capable of screening compounds for binding activity to
the proteins. Several methods of automating assays have been developed in
recent years so as to permit screening of tens of thousands of compounds
in a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773,
and other descriptions of chemical diversity libraries, which describe
means for testing of binding affinity by a plurality of compounds. The
development of suitable assays can be greatly facilitated by the
availability of large amounts of purified, soluble CX3Ckine as provided
by this invention.

[0134]For example, antagonists can normally be found once the protein has
been structurally defined. Testing of potential protein analogs is now
possible upon the development of highly automated assay methods using a
purified receptor. In particular, new agonists and antagonists will be
discovered by using screening techniques described herein. Of particular
importance are compounds found to have a combined binding affinity for
multiple CX3Ckine receptors, e.g., compounds which can serve as
antagonists for species variants of a CX3Ckine.

[0135]This invention is particularly useful for screening compounds by
using recombinant protein in a variety of drug screening techniques. The
advantages of using a recombinant protein in screening for specific
ligands include: (a) improved renewable source of the CX3Ckine from a
specific source; (b) potentially greater number of ligands per cell
giving better signal to noise ratio in assays; and (c) species variant
specificity (theoretically giving greater biological and disease
specificity).

[0136]One method of drug screening utilizes eukaryotic or prokaryotic host
cells which are stably transformed with recombinant DNA molecules
expressing a CX3Ckine receptor. Cells may be isolated which express a
receptor in isolation from any others. Such cells, either in viable or
fixed form, can be used for standard ligand/receptor binding assays. See
also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)
Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods
to detect cellular responses. Competitive assays are particularly useful,
where the cells (source of CX3Ckine) are contacted and incubated with a
labeled receptor or antibody having known binding affinity to the ligand,
such as 125I-antibody, and a test sample whose binding affinity to
the binding composition is being measured. The bound and free labeled
binding compositions are then separated to assess the degree of ligand
binding. The amount of test compound bound is inversely proportional to
the amount of labeled receptor binding to the known source. Any one of
numerous techniques can be used to separate bound from free ligand to
assess the degree of ligand binding. This separation step could typically
involve a procedure such as adhesion to filters followed by washing,
adhesion to plastic followed by washing, or centrifugation of the cell
membranes. Viable cells could also be used to screen for the effects of
drugs on CX3Ckine mediated functions, e.g., second messenger levels,
i.e., Ca++; cell proliferation; inositol phosphate pool changes; and
others. Some detection methods allow for elimination of a separation
step, e.g., a proximity sensitive detection system. Calcium sensitive
dyes will be useful for detecting Ca++ levels, with a fluorimeter or
a fluorescence cell sorting apparatus.

[0137]Another method utilizes membranes from transformed eukaryotic or
prokaryotic host cells as the source of a CX3Ckine. These cells are
stably transformed with DNA vectors directing the expression of a
CX3Ckine, e.g., an engineered membrane bound form. Essentially, the
membranes would be prepared from the cells and used in a receptor/ligand
binding assay such as the competitive assay set forth above.

[0138]Still another approach is to use solubilized, unpurified or
solubilized, purified CX3Ckine from transformed eukaryotic or prokaryotic
host cells. This allows for a "molecular" binding assay with the
advantages of increased specificity, the ability to automate, and high
drug test throughput.

[0139]Another technique for drug screening involves an approach which
provides high throughput screening for compounds having suitable binding
affinity to a CX3Ckine antibody and is described in detail in Geysen,
European Patent Application 84/03564, published on Sep. 13, 1984. First,
large numbers of different small peptide test compounds are synthesized
on a solid substrate, e.g., plastic pins or some other appropriate
surface, see Fodor, et al., supra. Then all the pins are reacted with
solubilized, unpurified or solubilized, purified CX3Ckine antibody, and
washed. The next step involves detecting bound CX3Ckine antibody.

[0140]Rational drug design may also be based upon structural studies of
the molecular shapes of the CX3Ckine and other effectors or analogs. See,
e.g., Methods in Enzymology vols 202 and 203. Effectors may be other
proteins which mediate other functions in response to ligand binding, or
other proteins which normally interact with the receptor. One means for
determining which sites interact with specific other proteins is a
physical structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which amino
acid residues form molecular contact regions. For a detailed description
of protein structural determination, see, e.g., Blundell and Johnson
(1976) Protein Crystallography Academic Press, NY.

[0141]A purified CX3Ckine can be coated directly onto plates for use in
the aforementioned drug screening techniques. However, non-neutralizing
antibodies to these ligands can be used as capture antibodies to
immobilize the respective ligand on the solid phase.

XI. Kits

[0142]This invention also contemplates use of CX3Ckine proteins, fragments
thereof, peptides, and their fusion products in a variety of diagnostic
kits and methods for detecting the presence of CX3Ckine or a CX3Ckine
receptor. Typically the kit will have a compartment containing either a
defined CX3Ckine peptide or gene segment or a reagent which recognizes
one or the other, e.g., receptor fragments or antibodies.

[0143]A kit for determining the binding affinity of a test compound to a
CX3Ckine would typically comprise a test compound; a labeled compound,
e.g., a receptor or antibody having known binding affinity for the
CX3Ckine; a source of CX3Ckine (naturally occurring or recombinant); and
a means for separating bound from free labeled compound, such as a solid
phase for immobilizing the CX3Ckine. Once compounds are screened, those
having suitable binding affinity to the CX3Ckine can be evaluated in
suitable biological assays, as are well known in the art, to determine
whether they act as agonists or antagonists to the receptor. The
availability of recombinant CX3Ckine polypeptides also provide well
defined standards for calibrating such assays.

[0144]A preferred kit for determining the concentration of, for example, a
CX3Ckine in a sample would typically comprise a labeled compound, e.g.,
receptor or antibody, having known binding affinity for the CX3Ckine, a
source of CX3Ckine (naturally occurring or recombinant), and a means for
separating the bound from free labeled compound, for example, a solid
phase for immobilizing the CX3Ckine. Compartments containing reagents,
and instructions, will normally be provided.

[0145]Antibodies, including antigen binding fragments, specific for the
CX3Ckine or ligand fragments are useful in diagnostic applications to
detect the presence of elevated levels of CX3Ckine and/or its fragments.
Such may allow diagnosis of the amounts of differently processed forms of
the CX3Ckine, e.g., successively degraded stalk structure. Such
diagnostic assays can employ lysates, live cells, fixed cells,
immunofluorescence, cell cultures, body fluids, and further can involve
the detection of antigens related to the ligand in serum, or the like.
Diagnostic assays may be homogeneous (without a separation step between
free reagent and antigen-CX3Ckine complex) or heterogeneous (with a
separation step). Various commercial assays exist, such as
radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), enzyme
immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA), and the like. For
example, unlabeled antibodies can be employed by using a second antibody
which is labeled and which recognizes the antibody to a CX3Ckine or to a
particular fragment thereof. Similar assays have also been extensively
discussed in the literature. See, e.g., Harlow and Lane (1988)
Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987)
Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price and
Newman (eds.) (1991) Principles and Practice of Immunoassay Stockton
Press, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press, NY.

[0146]Anti-idiotypic antibodies may have similar use to diagnose presence
of antibodies against a CX3Ckine, as such may be diagnostic of various
abnormal states. For example, overproduction of CX3Ckine may result in
production of various immunological or other medical reactions which may
be diagnostic of abnormal physiological states, e.g., in cell growth,
activation, or differentiation.

[0147]Frequently, the reagents for diagnostic assays are supplied in kits,
so as to optimize the sensitivity of the assay. For the subject
invention, depending upon the nature of the assay, the protocol, and the
label, either labeled or unlabeled antibody or receptor, or labeled
CX3Ckine is provided. This is usually in conjunction with other
additives, such as buffers, stabilizers, materials necessary for signal
production such as substrates for enzymes, and the like. Preferably, the
kit will also contain instructions for proper use and disposal of the
contents after use. Typically the kit has compartments for each useful
reagent. Desirably, the reagents are provided as a dry lyophilized
powder, where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay.

[0148]Many of the aforementioned constituents of the drug screening and
the diagnostic assays may be used without modification, or may be
modified in a variety of ways. For example, labeling may be achieved by
covalently or non-covalently joining a moiety which directly or
indirectly

provides a detectable signal. In any of these assays, theprotein, test
compound, CX3Ckine, or antibodies thereto can be labeled either directly
or indirectly. Possibilities for direct labeling include label groups:
radiolabels such as 125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.
No. 3,940,475) capable of monitoring the change in fluorescence
intensity, wavelength shift, or fluorescence polarization. Possibilities
for indirect labeling include biotinylation of one constituent followed
by binding to avidin coupled to one of the above label groups.

[0149]There are also numerous methods of separating the bound from the
free ligand, or alternatively the bound from the free test compound. The
CX3Ckine can be immobilized on various matrices followed by washing.
Suitable matrices include plastic such as an ELISA plate, filters, and
beads. Methods of immobilizing the CX3Ckine to a matrix include, without
limitation, direct adhesion to plastic, use of a capture antibody,
chemical coupling, and biotin-avidin. The last step in this approach
involves the precipitation of ligand/receptor or ligand/antibody complex
by any of several methods including those utilizing, e.g., an organic
solvent such as polyethylene glycol or a salt such as ammonium sulfate.
Other suitable separation techniques include, without limitation, the
fluorescein antibody magnetizable particle method described in Rattle, et
al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic
particle separation as described in U.S. Pat. No. 4,659,678.

[0150]Methods for linking proteins or their fragments to the various
labels have been extensively reported in the literature and do not
require detailed discussion here. Many of the techniques involve the use
of activated carboxyl groups either through the use of carbodiimide or
active esters to form peptide bonds, the formation of thioethers by
reaction of a mercapto group with an activated halogen such as
chloroacetyl, or an activated olefin such as maleimide, for linkage, or
the like. Fusion proteins will also find use in these applications.

[0151]Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence of a
CX3Ckine. These sequences can be used as probes for detecting levels of
the CX3Ckine message in samples from natural sources, or patients
suspected of having an abnormal condition, e.g., cancer or developmental
problem. The preparation of both RNA and DNA nucleotide sequences, the
labeling of the sequences, and the preferred size of the sequences has
received ample description and discussion in the literature. Normally an
oligonucleotide probe should have at least about 14 nucleotides, usually
at least about 18 nucleotides, and the polynucleotide probes may be up to
several kilobases. Various labels may be employed, most commonly
radionuclides, particularly 32P. However, other techniques may also
be employed, such as using biotin modified nucleotides for introduction
into a polynucleotide. The biotin then serves as the site for binding to
avidin or antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorophores, enzymes, or the like. Alternatively,
antibodies may be employed which can recognize specific duplexes,
including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or
DNA-protein duplexes. The antibodies in turn may be labeled and the assay
carried out where the duplex is bound to a surface, so that upon the
formation of duplex on the surface, the presence of antibody bound to the
duplex can be detected. The use of probes to the novel anti-sense RNA may
be carried out using many conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational probing, hybrid
released translation (HRT), and hybrid arrested translation (HART). This
also includes amplification techniques such as polymerase chain reaction
(PCR).

[0152]Diagnostic kits which also test for the qualitative or quantitative
presence of other markers are also contemplated. Diagnosis or prognosis
may depend on the combination of multiple indications used as markers.
Thus, kits may test for combinations of markers. See, e.g., Viallet, et
al. (1989) Progress in Growth Factor Res. 1:89-97.

XII. Receptor Isolation

[0153]Having isolated a binding partner of a specific interaction, methods
exist for isolating the counter-partner. See, Gearing, et al. (1989) EMBO
J. 8:3667-3676. For example, means to label a CX3Ckine without
interfering with the binding to its receptor can be determined. For
example, an affinity label or epitope tag can be fused to either the
amino- or carboxyl-terminus of the ligand. An expression library can be
screened for specific binding of the CX3Ckine, e.g., by cell sorting, or
other screening to detect subpopulations which express such a binding
component. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA
90:11267-11271. Alternatively, a panning method may be used. See, e.g.,
Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369. A
two-hybrid selection system may also be applied making appropriate
constructs with the available BAS-1 sequences. See, e.g., Fields and Song
(1989) Nature 340:245-246.

[0154]Protein cross-linking techniques with label can be applied to
isolate binding partners of a CX3Ckine. This would allow identification
of proteins which specifically interact with a CX3Ckine, e.g., in a
ligand-receptor like manner. Typically, the chemokine family binds to
receptors of the seven transmembrane receptor family, and the receptor
for the CX3Ckine is likely to exhibit a similar structure. Thus, it is
likely that the receptor will be found by expression in a system which is
capable of expressing such a membrane protein in a form capable of
exhibiting ligand binding capability.

[0155]The broad scope of this invention is best understood with reference
to the following examples, which are not intended to limit the invention
to specific embodiments.

[0159]A clone encoding the human CX3Ckine is isolated from a natural
source by many different possible methods. Given the sequences provided
herein, PCR primers or hybridization probes are selected and/or
constructed to isolate either genomic DNA segments or cDNA reverse
transcripts. Appropriate cell sources include human tissues, e.g., brain
libraries. Tissue distribution below also suggests source tissues.
Genetic and polymorphic or allelic variants are isolated by screening a
population of individuals.

[0160]PCR based detection is performed by standard methods, preferably
using primers from opposite ends of the coding sequence, but flanking
segments might be selected for specific purposes.

[0161]Alternatively, hybridization probes are selected. Particular AT or
GC contents of probes are selected depending upon the expected homology
and mismatching expected. Appropriate stringency conditions are selected
to balance an appropriate positive signal to background ratio. Successive
washing steps are used to collect clones of greater homology.

[0162]Further clones are isolated using an antibody based selection
procedure. Standard expression cloning methods are applied including,
e.g., FACS staining of membrane associated expression product. The
antibodies are used to identify clones producing a recognized protein.
Alternatively, antibodies are used to purify a CX3C chemokine, with
protein sequencing and standard means to isolate a gene encoding that
protein.

[0163]Genomic sequence based methods will also allow for identification of
sequences naturally available, or otherwise, which exhibit homology to
the provided sequences. Similar procedures will allow isolation of other
primate genes.

III. Isolation of Rodent CX3Ckine Clone

[0164]Similar methods are used as above to isolate an appropriate mouse
CX3C chemokine gene. Similar source materials as indicated above are used
to isolate natural genes, including genetic, polymorphic, allelic, or
strain variants. Species variants are also isolated using similar
methods, e.g., from rats, moles, muskrats, copybaras, etc.

IV. Isolation of an Avian CX3Ckine Clone

[0165]An appropriate avian source is selected as above. Similar methods
are utilized to isolate a species variant, though the level of similarity
will typically be lower for avian CX3C chemokine as compared to a human
to mouse sequence.

V. Expression; Purification; Characterization

[0166]With an appropriate clone from above, the coding sequence is
inserted into an appropriate expression vector. This may be in a vector
specifically selected for a prokaryote, yeast, insect, or higher
vertebrate, e.g., mammalian expression system. Standard methods are
applied to produce the gene product, preferably as a soluble secreted
molecule, but will, in certain instances, also be made as an
intracellular protein. Intracellular proteins typically require cell
lysis to recover the protein, and insoluble inclusion bodies are a common
starting material for further purification.

[0167]With a clone encoding a vertebrate CX3C chemokine, recombinant
production means are used, although natural forms may be purified from
appropriate sources. The protein product is purified by standard methods
of protein purification, in certain cases, e.g., coupled with
immunoaffinity methods. Immunoaffinity methods are used either as a
purification step, as described above, or as a detection assay to
determine the separation properties of the protein.

[0168]Preferably, the protein is secreted into the medium, and the soluble
product is purified from the medium in a soluble form. Alternatively, as
described above, inclusion bodies from prokaryotic expression systems are
a useful source of material. Typically, the insoluble protein is
solubilized from the inclusion bodies and refolded using standard
methods. Purification methods are developed as described above.

[0169]In certain embodiments, the protein is made in a eukaryotic cell
which glycosylates the protein normally. The purification methods may be
affected thereby, as may biological activities. The intact protein can be
processed to release the chemokine domain, probably due to a protease
cleavage event somewhere in the glycosylated stalk region close to the
chemokine/stalk boundary. While recombinant protein appears to be
processed, the physiological processes which normally do such in native
cells remain to be determined.

[0170]The product of the purification method described above is
characterized to determine many structural features. Standard physical
methods are applied, e.g., amino acid analysis and protein sequencing.
The resulting protein is subjected to CD spectroscopy and other
spectroscopic methods, e.g., NMR, ESR, mass spectroscopy, etc. The
product is characterized to determine its molecular form and size, e.g.,
using gel chromatography and similar techniques. Understanding of the
chromatographic properties will lead to more gentle or efficient
purification methods.

[0171]CX3C chemokine protein biochemistry was assessed in mammalian
expression systems. Human embryonic kidney 293 cells (HEK 293)
transfected with a mammalian expression construct encoding full-length
CX3C chemokine were metabolically labeled with 35S cysteine and
methionine. CX3C chemokine was produced as a protein of Mr ˜95 kDa;
control transfected supernatants contained no such species. Neuraminidase
and gycosidases reduced the Mr of CX3C chemokine from ˜95 kDa to
˜45 kDa, suggesting that the recombinant form is glycosylated
substantially. Thus CX3C chemokine cDNA, encoding a predicted
membrane-bound protein, encodes a glycoprotein which is released from
cells by an undefined mechanism.

[0174]Polyclonal serum is raised against a purified antigen, purified as
indicated above, or using synthetic peptides. A series of overlapping
synthetic peptides which encompass all of the full length sequence, if
presented to an animal, will produce serum recognizing most linear
epitopes on the protein. Such an antiserum is used to affinity purify
protein, which is, in turn, used to introduce intact full length protein
into another animal to produce another antiserum preparation.

[0176]Distribution of the protein or gene products are determined, e.g.,
using immunohistochemistry with an antibody reagent, as produced above,
or by screening for nucleic acids encoding the chemokine. Either
hybridization or PCR methods are used to detect DNA, cDNA, or message
content. Histochemistry allows determination of the specific cell types
within a tissue which express higher or lower levels of message or DNA.
Antibody techniques are useful to quantitate protein in a biological
sample, including a liquid or tissue sample. Immunoassays are developed
to quantitate protein.

[0177]Hybridization techniques were applied to the tissue types in Table 3
with positive or negative results, as indicated. The commercial tissue
blots may have cellular contamination from resident cells, e.g., from
blood or other cells which populate the tissue. The large and small
transcripts correspond to sizes about 4 kb and less than about 2 kb,
respectively.

[0180]Since the endothelium is a major site of chemokine action, a
northern blot was performed to ascertain if CX3Ckine was expressed in
this tissue. Human CX3Ckine was also shown to be expressed on human
activated primary endothelial cells by both mRNA and protein expression.
This suggests that CX3Ckine may be involved in leukocyte trafficking in
various organs.

[0183]The CX3C chemokine gene has been mapped to human chromosome 16. A
BIOS Laboratories (New Haven, Conn.) mouse somatic cell hybrid panel was
combined with PCR. These mapping studies also indicate the possibility of
a pseudogene or related gene on human chromosome 14. Sequencing of
genomic DNA fragments suggests CX3C chemokine gene has an intron which
begins near or in the codon encoding Ile 64. Other intron/exon boundaries
have yet to be mapped. This location is distinct from the chromosomal
mapping locations of the other C, CC, or CXC chemokine families,
consistent with the CX3Ckine being a separate gene family within the
chemokines.

X. Biological Activities, Direct and Indirect

[0184]The 293 human embryonic kidney cell line (HER 293) was transfected
with either the membrane bound form of human CX3Ckine (293-CX3Ckine), the
chemokine domain plus the "stalk" region, or a control vector without an
insert. The transfected cells were subsequently cultured with either
monocytes, T cells, or peripheral mononuclear (PMN) cells to assay
relative adherence of these cells to CX3Ckine. Specifically,
5×104 cells per well of HEK 293 transfected cells were seeded
in a 96 well plate. 2×105 monocytes, T cells, or PMNs,
metabolically labeled with 35S-methionine and cysteine (Amersham,
Arlington Heights, Ill.), were added to each well. The plate was then
incubated at 37° C. for various time points. The wells were washed
2 times RPMI supplemented with 1% FCS. Plates were then read in a
Millipore Cytofluor at 485/530 nm.

[0185]In all cases, adherence to HEK 293 cells transfected with the
membrane bound form of CX3Ckine was significantly enhanced when compared
to the truncated CX3Ckine or mock transfected cells. Interestingly, only
the membrane bound form possessed this proadhesive activity, leading to
the conclusion that CX3Ckine, in its membrane bound form, may serve as a
regulator of circulating leukocytes.

[0186]In another experiment, the recombinant soluble form of the chemokine
domain of CX3Ckine (rCx3C) was added to HEK 293-CX3C cells and monocytes
at a concentration of 1 μM per well, and assayed as described above.
rCX3C was able to antagonize adhesion of monocytes to HEK 293-CX3C cells.
A similar experiment was performed to investigate the effect on T cell
adherence. Comparable results were obtained. Thus rCX3C may function as a
negative regulator of circulating leukocytes.

[0187]A comparison of three different forms of human CX3Ckine was
performed to analyze variations in chemoattractant activity that may be
due to the structure of CX3Ckine. CX3C 1.7 (chemokine domain plus the
entire stalk region), CX3C 0.7 (chemokine domain plus one-half stalk
region), and CX3C CK (chemokine domain only) were subjected to the
chemotaxicity assay described above, their ability to attract T cells was
analyzed. CX3C 1.7 displayed a slightly better dose dependent ability to
attract T cells relative to the other forms of CX3Ckine.

[0188]A robust and sensitive assay is selected as described above, e.g.,
on immune cells, neuronal cells, or stem cells. Chemokine is added to the
assay in increasing doses to see if a dose response is detected. For
example, in a proliferation assay, cells are plated out in plates.
Appropriate culture medium is provided, and chemokine is added to the
cells in varying amounts. Growth is monitored over a period of time which
will detect either a direct effect on the cells, or an indirect effect of
the chemokine.

[0190]Other assays will be those which have been demonstrated with other
chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in
Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &
Immunol. 109:97-109. Effects of truncated stalk structures will be
similarly evaluated.

XI. Structure Activity Relationship

[0191]Information on the criticality of particular residues is determined
using standard procedures and analysis. Standard mutagenesis analysis is
performed, e.g., by generating many different variants at determined
positions, e.g., at the positions identified above, and evaluating
biological activities of the variants. This may be performed to the
extent of determining positions which modify activity, or to focus on
specific positions to determine the residues which can be substituted to
either retain, block, or modulate biological activity.

[0192]Alternatively, analysis of natural variants can indicate what
positions tolerate natural mutations. This may result from populational
analysis of variation among individuals, or across strains or species.

Samples from selected individuals are analysed, e.g., by PCR analysis and
sequencing. This allows evaluation of population polymorphisms.
Particularly, as described above, many of the biological activities of
the chemokine domain attached to different portions or extents of the
stalk structure may result.

XII. Screening for Agonists/Antagonists

[0193]Agonists or antagonists are screened for ability to induce or block
biological activity. The candidate compounds, e.g, sequence variants of
natural CX3Ckines, are assayed for their biological activities.
Alternatively, compounds are screened, alone or in combinations, to
determine effects on biological activity.

XIII. Isolation of a Receptor for CX3C Chemokine

[0194]Based on the proadherent properties of CX3Ckine, 7 transmembrane
G-protein receptor was found to be expressed by monocytes and T cells. It
was also discovered that the chemokine domain is the only region of
CX3Ckine that can engage the receptor. Binding assays with known
chemokine receptor revealed that CX3Ckine does not engage chemokine
receptors CCR 1 through 5, CXCR 1 and 2, or the Duffy antigen receptor .
. . CX3Ckine can, however, bind to a virally encoded chemokine receptor,
CMV-US28.

[0195]Alternatively, CX3C chemokine can be used as a specific binding
reagent to identify its binding partner, by taking advantage of its
specificity of binding, much like an antibody would be used. A binding
reagent is either labeled as described above, e.g., fluorescence or
otherwise, or immobilized to a substrate for panning methods. The typical
chemokine receptor is a seven transmembrane receptor.

[0196]The purified protein is also be used to identify other binding
partners of CX3Ckine as described, e.g., in Fields and Song (1989) Nature
340:245-246.

[0197]The binding composition, e.g., chemokine, is used to screen an
expression library made from a cell line which expresses a binding
partner, i.e. receptor. Standard staining techniques are used to detect
or sort intracellular or surface expressed receptor, or surface
expressing transformed cells are screened by panning. Screening of
intracellular expression is performed by various staining or
immunofluorescence procedures. See also McMahan, et al. (1991) EMBO J.
10:2821-2832.

[0198]For example, on day 0, precoat 2-chamber permanox slides with 1 ml
per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at room
temperature. Rinse once with PBS. Then plate COS cells at
2-3×105 cells per chamber in 1.5 ml of growth media. Incubate
overnight at 37° C.

[0199]On day 1 for each sample, prepare 0.5 ml of a solution of 66
μg/ml DEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum
free DME. For each set, a positive control is prepared, e.g., of human
CX3C chemokine cDNA at 1 and 1/200 dilution, and a negative mock. Rinse
cells with serum free DME. Add the DNA solution and incubate 5 hr at
37° C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5
min. Remove and wash once with DME. Add 1.5 ml growth medium and incubate
overnight.

[0200]On day 2, change the medium. On days 3 or 4, the cells are fixed and
stained. Rinse the cells twice with Hank's Buffered Saline Solution
(HBSS) and fix in 4% paraformaldehyde (PEA)/glucose for 5 min. Wash
3× with HBSS. The slides may be stored at -80° C. after all
liquid is removed. For each chamber, 0.5 ml incubations are performed as
follows. Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN3 for 20
min. Cells are then washed with HESS/saponin 1×. Add chemokine or
chemokine/antibody complex to cells and incubate for 30 min. Wash cells
twice with HBSS/saponin. If appropriate, add first antibody for 30 min.
Add second antibody, e.g., Vector anti-mouse antibody, at 1/200 dilution,
and incubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABC
horseradish peroxidase solution, and preincubate for 30 min. Use, e.g., 1
drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml
HBSS/saponin. Wash cells twice with HESS/saponin. Add ABC HRP solution
and incubate for 30 min. Wash cells twice with HBSS, second wash for 2
min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5
to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of
H2O2 per 5 ml of glass distilled water. Carefully remove
chamber and rinse slide in water. Air dry for a few minutes, then add 1
drop of Crystal Mount and a cover slip. Bake for 5 min at 85-90°
C.

[0201]Evaluate positive staining of pools and progressively subclone to
isolation of single genes responsible for the binding.

[0203]Another strategy is to screen for a membrane bound receptor by
panning. The receptor cDNA is constructed as described above. The ligand
can be immobilized and used to immobilize expressing cells.
Immobilization may be achieved by use of appropriate antibodies which
recognize, e.g., a FLAG sequence of a chemokine fusion construct, or by
use of antibodies raised against the first antibodies. Recursive cycles
of selection and amplification lead to enrichment of appropriate clones
and eventual isolation of receptor expressing clones.

[0205]All references cited herein are incorporated herein by reference to
the same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.

[0206]Many modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to those
skilled in the art. The specific embodiments described herein are offered
by way of example only, and the invention is to be limited only by the
terms of the appended claims, along with the full scope of equivalents to
which such claims are entitled.